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Cellular and Molecular Endocrinology Group, Biomedical Sciences Division, King’s College London, Campden Hill Road, Kensington, London, W8 7AH, United Kingdom
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Abstract |
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 Top Abstract I. IntroductionII. Protein Kinases in...III. Phosphorylation of...IV. Protein Kinases and...V. Protein Phosphatases and...VI. Protein Kinase-Independent...VII. Summary and Future...References |
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I. Introduction |
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TopAbstractI. IntroductionII. Protein Kinases in...III. Phosphorylation of...IV. Protein Kinases and...V. Protein Phosphatases and...VI. Protein Kinase-Independent...VII. Summary and Future...References |
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The mechanisms through which ß-cells recognize nutrient and nonnutrient stimuli have been studied in detail. These mechanisms and their relative importance in the generation and maintenance of physiologically relevant secretory responses have been reviewed extensively elsewhere (5, 6, 7, 8). In general, both classes of secretagogues control the secretory process by modifying the concentration or availability of a number of intracellular regulators within ß-cells, including Ca2+, cyclic nucleotides, and products of phospholipid hydrolysis. Over the past few years, considerable advances have been made in our understanding of the mechanisms controlling the generation and actions of intracellular regulators within ß-cells, and this subject has been the topic of some excellent and comprehensive reviews (7, 9, 10, 11, 12, 13, 14, 15). In brief, it is now generally accepted that the metabolism of glucose and other nutrient secretagogues within the ß-cell results in the closure of ATP-sensitive K+ (KATP) channels in the plasma membrane, leading to depolarization of the cell, with a consequent influx of extracellular Ca2+ through voltage-sensitive channels (reviewed in Ref. 16). Although useful, this electrophysiological model does not fully describe ß-cell secretory responses to nutrients. Not all of the effects of nutrient secretagogues on insulin secretion can be explained by closure of KATP channels (17, 18), and it is important to note that nutrients also elevate intracellular concentrations of cAMP, and of regulators derived from membrane phospholipids, including inositol trisphosphate (IP3), diacylglycerols (DAG), arachidonic acid (AA), and phosphatidic acid. Evidence is also accumulating for Ca2+-independent mechanisms through which ß-cells recognize and respond to glucose (19), although it is still too early to determine the physiological importance of this pathway(s).
Despite the differences in their mechanisms of recognition by
ß-cells, receptor-mediated, nonnutrient insulin secretagogues
influence the secretory process through the same intracellular
regulators as do nutrient secretagogues. For example, cholinergic
muscarinic agonists, such as carbachol (CCh), stimulate ß-cell
depolarization and Ca2+ influx (20); CCh and peptide
hormones such as bombesin, cholecystokinin (CCK), and arginine
vasopressin (AVP) activate phospholipase C (PLC) with the subsequent
generation of IP3 and DAG from inositol phospholipids
(21, 22, 23, 24, 25); CCh also activates phospholipase A2
(PLA2), which hydrolyzes phosphatidylcholine to generate AA
and lysophospholipids (26); glucagon, glucose-dependent insulinotropic
polypeptide (GIP), and pituitary adenylate cyclase (AC)-activating
polypeptide (PACAP) stimulate AC to produce elevations in ß-cell cAMP
(27, 28, 29), whereas 
2-adrenoreceptor agonists,
somatostatin and galanin, inhibit AC and reduce intracellular cAMP
(reviewed in Ref. 30).
Although these intracellular regulators have been implicated in the
control of insulin secretion, the mechanisms through which changes in
their concentrations produce a controlled secretory response are not
yet well understood. In this review we will examine whether the ability
of the intracellular regulators to control the activity of protein
kinases, and thus the phosphorylation state (and function) of
intracellular proteins, could provide a common transduction mechanism
for these diverse signals. This has been an area of sporadically active
ß-cell research since the early 1970s (31), but one that is
singularly lacking in comprehensive reviews. For this reason, our
coverage of the topic has been deliberately broad. We first review the
available information on the occurrence and characteristics of
regulated protein kinases in pancreatic ß-cells (Section
II and Table 1
) and compile the
disparate data on the protein substrates for the kinases in ß-cells
(Section III and Table 2).
We then consider the evidence for and against the involvement of
particular kinase and phosphatase activities in insulin-secretory
responses (Sections IV and V) and discuss the hypothesis
that protein kinase activation is an essential step in
stimulus-secretion coupling in ß-cells.
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II. Protein Kinases in ß-Cells: Expression and Characteristics |
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TopAbstractI. IntroductionII. Protein Kinases in...III. Phosphorylation of...IV. Protein Kinases and...V. Protein Phosphatases and...VI. Protein Kinase-Independent...VII. Summary and Future...References |
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The main purpose of the pancreatic ß-cell is to synthesize and
secrete insulin, so most attention has naturally been paid to those
kinases that may be involved in the insulin-secretory process. Since
little is yet known about the substrate specificity of these kinases in
ß-cells, it is common (and useful) to classify them on the basis of
their activators, rather than on the more biochemically correct basis
of their substrate specificity. Thus, several distinct classes of
protein kinases whose activities can be modified by Ca2+,
cyclic nucleotides, and products of phospholipid hydrolysis have been
identified both in islets of Langerhans and in insulin-secreting cell
lines, and these are summarized in Table 1
. The kinase activities that
have been identified in ß-cells to date are similar (or identical) to
enzymes that have been studied in much more detail in other tissues.
The expression, structure, and regulation of these enzymes have been
reviewed in detail elsewhere (35, 41, 42, 43, 44, 45, 46). The present review will
therefore consider only the available information about the occurrence
and characteristics of kinase activities in insulin-secreting tissues
and the physiological relevance of these enzymes to the control of
insulin secretion.
A. Ca2+/calmodulin-dependent protein kinases
A number of serine/threonine kinases are dependent upon the
presence of Ca2+ and the Ca2+-binding protein,
calmodulin (CaM), for their activation. Ca2+/CaM-dependent
protein kinase activity in hamster insulinoma cells has been shown to
phosphorylate endogenous substrates on serine and/or threonine residues
(47), and ß-cells are currently known to contain at least three types
of Ca2+/CaM-dependent protein kinase. Myosin light chain
kinase (MLCK) has been reported to be present in rat insulinoma cells
(48), HIT cells (49), and in islets isolated from rat (50) and human
(49, 51) pancreas. ß-cell MLCK has been identified as an enzyme that
catalyzes the phosphorylation of exogenous myosin light chains with
affinities for Ca2+ [affinity constant (Ka) =
10 µM], CaM (Ka = 2 nM), myosin
light chains [Michaelis-Menton constant (Km) = 70
µM], and ATP (Km = 70 µM), and
a susceptibility to inhibition by trifluoperazine [TFP, inhibition
constant (Ki) = 10 µM], which are similar to
those reported for MLCK isolated from other tissues. Two other
Ca2+/CaM-dependent protein kinases (CaMK), distinct from
MLCK, have been identified in rat islets (52, 53, 54, 55) and human islets (49, 51).
A study comparing islet CaMK activity with the multifunctional CaMK II enzyme purified from rat brain demonstrated that islet CaMK consists of two distinct activities, which have been identified as CaMK II and CaMK III (55). Of the three Ca2+/CaM-dependent kinase activities identified in ß-cells, the multifunctional CaMK II is most likely to subserve a role in ß-cell stimulus-secretion coupling, since both CaMK III and MLCK are thought to be dedicated to the regulation of a single function (42): MLCK preferentially phosphorylates myosin light chains, while the CaMK III activity in rat islets specifically phosphorylates elongation factor 2, a protein of 102 kDa molecular mass, which is not a substrate for other Ca2+/CaM kinases (55), as has been reported previously for brain CaMK III (56, 57).
Islet CaMK II and that found in MIN6 insulinoma cells is similar to,
and perhaps identical with, CaMK II purified from rat brain (55, 58),
and this enzyme is responsible for the CaMK activity that is associated
with a particulate (190,000 x g) fraction (49) that
has been identified as microsomal membranes (53, 54). Because of the
difficulties inherent in the purification to homogeneity of enzymes
from limited quantities of islets, little detailed information is
available about the physical characteristics of ß-cell CaMK II.
However, since it is known to be very similar to brain CaMK II (55),
islet CaMK II probably exists as a holoenzyme of approximately 550 kDa
molecular mass, consisting of a number (8, 9, 10) of subunits, each of
which contain both regulatory and catalytic activity. CaMK II
autophosphorylates on several serine and threonine residues in the
presence of Ca2+ and CaM (41), and the 53- to 55-kDa
substrates for CaMK II reported in many studies (see Table 2) are
likely to be subunits of the CaMK II holoenzyme, as discussed in
Section III.B. Four independent genes coding for -,
ß-, 
-, and 
- isoforms of CaMK II, and corresponding splice
variants, have been identified in a variety of tissues. All four
isoforms (, ß3, B, E,
and 2) have been detected in islets (59, 60, 61, 62), with the
-isoform reported to be localized to rat islet microsomes (61) and
the 2-isoform to ß-cell-secretory granules (62).
Intriguingly, there has been a report of the expression of a truncated
-isoform of CaMK II in human islets, which is likely to be regulated
in the same manner as the holoenzyme but to exist as a monomer because
it lacks an association domain (63). The functional significance of the
truncation has not been established, but the expression of the
truncated CaMK II may be important for targeting the enzyme (63).
B. Cyclic nucleotide-dependent protein kinases
cAMP-dependent protein kinase A (PKA) was first isolated
from rat and guinea-pig islets of Langerhans by Montague and Howell
(64, 65) and shown by gel filtration to be a protein of apparent
molecular mass 180 kDa which, in the presence of cAMP, dissociated into
a catalytic subunit of 75 kDa and a regulatory subunit of 90 kDa. The
PKA activity in extracts of rat islets was stimulated by prior
treatment of the islets with agents known to increase intracellular
cAMP, including glucagon and a variety of methylxanthine
phosphodiesterase inhibitors (66). This PKA activity was mainly
associated with a postmicrosomal supernatant fraction and had similar
characteristics to PKA activity in other tissues, with a Ka
for cAMP of 
0.01 µM, a Km for ATP of 11
µM, and a pH optimum of 6.2 (65). PKA activity in
homogenates of rat islets of Langerhans was confirmed as being found in
the 100,000 x g supernatant fluid (67), while PKA
activity in a hamster transplantable islet cell tumor was also found
predominantly (70–80%) in the postmicrosomal supernatant fraction and
consisted of a regulatory subunit of approximate molecular mass 90 kDa
and a catalytic subunit of 33 kDa (68). In contrast, the results of
other studies suggested that a significant proportion of islet PKA was
associated with either the plasma membrane (69) or with the secretory
granule fraction (70, 71), perhaps suggestive of a role in exocytosis.
Although these initial studies produced no evidence for the existence
of PKA isoforms in islets, a subsequent detailed study demonstrated
that two isoforms of PKA could be identified in extracts of rat islets
after separation by chromatography on
diethylaminoethyl-cellulose (72). The two isoforms found in rat
islets were identified as type I and type II PKA by their differential
susceptibility to dissociation into regulatory and catalytic subunits
(73). The PKA in rat islet homogenates had a calculated molecular mass
of 144 kDa, a Ka for cAMP of 0.08 µM, and a
Km for exogenous histone IIA of 0.08 mg/ml (72). The type I
and type II isoforms purified from rat islets on
diethylaminoethyl-cellulose had Km values for ATP of 16.1
µM and 15.4 µM, respectively (72). In a
later study, photoaffinity labeling of islet proteins using
[3H]azido-cAMP identified a cytosolic cAMP-binding
protein of apparent molecular mass 54 kDa (74). This protein was
tentatively identified as the regulatory subunit of type II PKA, since
it had an isoelectric point (pI) of 5.0–5.5, was itself a
substrate for cAMP-induced phosphorylation, and was found to be part of
a native complex of approximate molecular mass 180 kDa (74). There are
at least four different types of regulatory subunit (RI, RIß,
RII, RIIß) and three catalytic subunits (C, Cß, C) that
are expressed in a tissue-specific manner. To date, there is no
information about which isoforms of the regulatory and catalytic
subunits are expressed in ß-cells, although it seems likely that they
will express RI, RII, and C since these are found in all
tissues examined (43, 44). The subcellular localization of PKA is
dependent upon the type of R subunit in the holoenzyme. Both types of
RI subunit are primarily cytosolic, so type I PKA is a cytosolic
enzyme. In contrast, RII subunits can interact with specific binding
proteins located on intracellular membranes, and type II PKA can thus
be associated with plasma, nuclear, and secretory granule membranes
(44, 75).
It therefore seems likely that, in common with many other cell types, pancreatic ß-cells contain two isoforms of PKA, each of which comprises a holoenzyme of two regulatory (R) subunits and two catalytic (C) subunits. Under resting conditions the phosphorylating activity of the catalytic subunits is inhibited by association with the regulatory subunits. Increased availability of cAMP results in its binding to the regulatory subunits causing a dissociation of the holoenzyme into a R2·cAMP4 dimer and two free active catalytic subunits. A subsequent decrease in the concentration of cAMP will favor dissociation of cAMP from the regulatory subunits, the reassociation of regulatory and catalytic subunits, and the resultant inhibition of catalytic activity.
Cyclic GMP-dependent protein kinases (PKG) have been isolated from several mammalian tissues, although the level of PKG activity is generally much lower than that of PKA (76). Islets of Langerhans contain guanylate cyclase and cyclic GMP (77), but the regulation of guanylate cyclase by insulin secretagogues and the importance of cyclic GMP in the control of insulin secretion are matters of some debate (see Section IV.B). Whatever signaling function cyclic GMP serves in islets, it may act through protein phosphorylation since a PKG has been identified in rat islets on the basis of cyclic GMP-dependent phosphorylation of exogenous arginine-rich histone (78). Separation of islet cyclic nucleotide-dependent kinase activities on Sephadex G-200 columns produced three peaks of enzyme activity, one of which had a much greater affinity for cyclic GMP than for cAMP (Ka values of 0.05 µM and 1.2 µM, respectively) (78). Our measurements of cyclic GMP-dependent phosphorylation by ß-cell extracts demonstrated a component that was not inhibited by the PKA-selective inhibitor, PKI6–22, and that we therefore attribute to PKG activity. The PKG activity in these experiments was 2.9 ± 0.04 pmol/106 cells/min, and represented 9.3% of the total cyclic nucleotide-dependent kinase activity in the ß-cell extracts (our unpublished results).
C. Ca2+/phospholipid-dependent protein kinases
The family of Ca2+/phospholipid-dependent protein
kinases known as protein kinase C (PKC) appears to be ubiquitous in
mammalian tissues (reviewed in Refs. 45, 79, 80), and is composed
of isoforms that can be classified into three groups: those that are
Ca2+-dependent (conventional isoforms: , ß, ),
those that are Ca2+-independent (novel isoforms: , 
,
, 
), and those that are Ca2+-independent and do not
bind DAG or phorbol esters (atypical isoforms: 
, 
/
, µ).
PKC in islets of Langerhans (81, 82) and in insulin-secreting cell
lines (82, 83) was originally identified as a
Ca2+-dependent protein kinase that required the presence of
an acidic phospholipid, such as phosphatidylserine (PS), for its
activation. The sensitivity of conventional isoforms of PKC to
activation by Ca2+ is greatly increased by the presence of
DAGs, or by tumor-promoting phorbol esters that substitute for DAG.
Studies using exogenous histone as a phosphorylation substrate for the
overall activities of Ca2+/DAG-sensitive PKC isoforms
expressed in insulin-secreting cells (82, 84) suggested that ß-cell
PKC has similar characteristics to those reported for other tissues
(see Refs. 79, 80 for references) with a Km for ATP of
10 µM, Ka for Ca2+ of 3.9–10
µM, Ka for PS of 12–18 µg/ml,
Ka for diolein of 2.5 µg/ml, and Km for
histone HI of 0.5 µM. PKC purified from rat islets also
shows a Ca2+-dependent activation in the presence of
micromolar concentrations of AA (85, 86) and a variety of other
cis-unsaturated fatty acids (87). Under unstimulated
conditions, DAG/PS/Ca2+-sensitive PKC activity is mainly
associated with a cytosolic fraction in islets (88, 89, 90) or
insulin-secreting cells (91, 92).
The activation of DAG-sensitive PKC isoforms is usually accompanied by
translocation, involving a redistribution of enzyme activity from a
predominantly cytosolic form to a membrane-associated form (93), and
there have been several reports of the translocation of activated PKC
in islets, ß-cells, and insulin-secreting cell lines
(e.g., Refs. 88, 89, 90, 91, 94, 95, 96, 97). There is little information
about the identity of the membrane compartments to which PKC isoforms
translocate upon activation in ß-cells. PKC activity has been
demonstrated to form a Ca2+-dependent, reversible
association with insulin-containing secretory granules (83), and
activated PKC- forms transient associations with the ß-cell
cytoskeleton (97). An immunocytochemical study suggested that glucose
stimulated the translocation of PKC- from the cytoplasm to a
periplasma membrane location (95), and a more recent study using
confocal fluorescence microscopy has demonstrated glucose-induced
redistribution of PKC- and - to the periphery of islet cells,
while PKC- and - translocated to perinuclear sites (98). Work in
other cell types suggests that the targeting of PKC isoforms to
particular membranes is due to the subcellular localization of specific
anchoring proteins known as receptors for activated C-kinase (RACKs)
(38). Nothing is yet known about RACK expression in ß-cells, although
peptides derived from the RACK binding sites of PKC isoforms are
reported to inhibit stimulus-dependent translocation of PKC isoforms in
rat islets (98). The kinase-anchoring protein interaction may offer
another target for the pharmacological modification of kinase function,
and thus of insulin secretion.
In many tissues the activation and translocation of PKC are followed by its proteolytic cleavage by membrane-associated, Ca2+-activated neutral proteases (99), and this mechanism is responsible for the down-regulation of cellular PKC activity by prolonged exposure to PKC-activating phorbol esters (reviewed in Ref. 80). Similar mechanisms appear to exist in pancreatic ß-cells since there have been several demonstrations that phorbol esters down-regulate PKC activity in islets (100, 101, 102, 103, 104) and insulin-secreting cell lines (91, 92), and that down-regulation of islet PKC can be inhibited by preventing the activation of Ca2+-activated neutral protease (88).
There have been many reports of expression of PKC isoforms in
ß-cells, but these have often been contradictory, which makes it
difficult to interpret much of the published data. Early studies
suggested that pancreatic ß-cells contained the -isoform (105) and
the ßII-isoform (106), but neither the ßI-isoform nor the
-isoform, whereas rat insulinoma (RINr) cells contained the
-isoform, but not the ß-isoforms. It was subsequently reported
that rat islets contained both the -isoform and a ß-isoform of PKC
(94, 95, 107, 108, 109) although there was some disagreement about which
endocrine cells within the islets expressed particular PKC isoforms.
Thus, the -isoform was localized in either the ß-cells (95, 105, 107, 109) or in peripheral islet cells (108), while a ß-isoform was
found in ß-cells (106), in peripheral islet cells but not in
ß-cells (95, 108), or was not detectable in any islet cells (109).
There is general agreement that the -isoform of PKC is not found in
ß-cells, although there is a report that the -isoform is
detectable in glucagon-containing -cells (109). It has been reported
that the -isoform of PKC is found only in somatostatin-containing
-cells (109), but other studies have indicated that it was present
in a radiation-induced transplantable insulinoma (110), in MIN6
ß-cells (111), and in islets from ob/ob mice which contain
more than 90% ß-cells (112). Observations that the -isoform was
present in pancreatic islets, but not localized to a particular
endocrine cell type (108), have been extended by the report that PKC
was the predominant isoform in both insulinoma-derived ß-cells
and whole islets and was associated with the cytoskeleton (110). Two
recent reports have indicated that MIN6, RINm5F, and ßTC3 ß-cell
lines express the µ-isoform (111, 113), but it is not yet clear
whether this particular PKC species is expressed in ß-cells of normal
islets. The -isoform of PKC has been detected in islets and ß-cell
lines (108, 110, 111, 113, 114), as has a novel PKC isoform, (111, 113, 114), which shows 70% sequence homology with the -isoform.
Identification of particular isoforms of PKC in ß-cells has relied to
a large extent on immunological criteria, and some of the discrepancies
in the reported expression of PKC isoforms in islet cells may reflect
dubious specificities of the antisera used. For example, an early study
of PKC isoforms in rat islet homogenates identified an -isoform of
PKC but found that the ß-isoform immunoreactivity migrated on
polyacrylamide gels with an apparent molecular mass of 50 kDa, rather
than the expected 80 kDa (94). However, a subsequent report from the
same group, but using different antisera, detected both - and
ß-isoform immunoreactivities migrating with apparent molecular masses
of 80 kDa (95). A further cause of conflicting results may stem from
changes in the expression of PKC isoforms during development, since it
has been reported that the expression of PKC isoforms differs in islets
from fetal, neonatal, and adult rats (109). Some of the uncertainty has
been resolved by a recent comprehensive study of the isoforms expressed
in MIN6 ß-cells: a combination of RT-PCR, Northern blotting, and
immunoblotting methodologies indicated the presence of PKC-, -
ßII, -, -, -, -(), and -µ isoforms (111), although
whether or not MIN6 insulinoma cells offer an accurate reflection of
PKC isoform expression in authentic ß-cells remains to be seen.
Furthermore, it is a distinct possibility that there are interspecies
variations in isoform expression, and it is not clear whether murine
ß-cells are a good model for nonrodent species.
D. Mitogen-activated protein kinases (MAPKs)
The MAPK family of proteins are a distinct group of kinases that
are activated by dual phosphorylation on threonine and tyrosine
residues. They comprise parallel signaling cascades, which appear to be
ubiquitous in mammalian cells, and include p42/44 MAP kinases, p38
reactivating kinase (RK), and stress-activated protein kinases (SAP
kinases). There have been many recent reviews on the structure and
regulation of the MAPK family of kinases (e.g., Refs. 115, 116).
The expression and function in ß-cells of MAPKs have received
relatively little attention to date, and the isoforms that are
expressed in islets and ß-cell lines are summarized in Table 1. Our
studies have shown that two forms of MAPK (42 and 44 kDa) are expressed
in rodent islets and in MIN6 ß-cells (117), and the 44-kDa isoform of
MAPK has also been identified in INS-1 cells, another ß-cell line
(118). Rather than being activated directly by second messengers, MAPK
activities are regulated by another kinase(s) (MAPK kinase, also known
as MEK; 45–46 kDa) which phosphorylates MAPKs on tyrosine and
threonine residues (reviewed in Ref. 119). MEK may be phosphorylated
and activated by the upstream serine/threonine kinases, MEK kinase
(MEKK), and Raf-1, and we have recently identified both of these
proteins in ß-cells by immunoblotting (120). There is currently
little information about the expression or activities of RK or SAP
kinases in insulin-secreting cells.
The identities and functions of ß-cell MAPK substrates have not been established, although there is some evidence that induction of early response genes (junB, nur77, and zif268) in INS-1 cells is associated with MAPK activation (118). In other tissues, cytosolic PLA2 (cPLA2) is phosphorylated and activated by p42 MAP kinase (121, 122), but glucose-induced cPLA2 phosphorylation in neonatal islets is reported to be independent from the activation of MAPK (123).
E. Protein tyrosine kinases
It is now well established that kinases that phosphorylate their
substrates on tyrosine residues (protein tyrosine kinases; PTKs) are
important in cellular regulation, and several families of PTK have been
identified in eukaryotic cells (see Refs. 33, 37). However, compared
with serine/threonine kinases, relatively little information is yet
available about the identities and possible functions of PTKs and PTK
substrates in the endocrine cells of the pancreas (see Tables 1 and 2).
PTK activity has been demonstrated in islets and in ß-cell lines
using antiphosphotyrosine antibodies to probe Western blots of islet
proteins (117, 124, 125, 126), or to immunoprecipitate
tyrosine-phosphorylated proteins from extracts (127, 128), and it is
clear that ß-cell proteins over a wide range of molecular masses are
substrates for PTKs. These proteins are also substrates for very active
phosphotyrosine phosphatase activities: under resting conditions the
phosphotyrosine content of ß-cell proteins is relatively low, but
this is rapidly and markedly enhanced by inhibition of phosphotyrosine
phosphatase activity, indicative of rapid
phosphorylation/dephosphorylation cycling of the substrates within the
cell. The tonic activity of tyrosine kinases under resting conditions
and the tight control of tyrosine phosphorylation exerted by
phosphotyrosine phosphatases might imply important physiological
functions within the ß-cell, although these have yet to be determined
(Section IV.E).
Several receptor and cytoplasmic PTKs in RINm5F cells, fetal rat
islets, and adult mouse islets have been identified by PCR
amplification of cDNAs using PTK-specific primers (129). A novel PTK
identified in RINm5F cells by PCR (120) has now been used to screen a
ß-cell cDNA library, and the amino acid sequence of a positive clone
indicated that it is a member of the src family of cytoplasmic PTKs
(130). The nonreceptor tyrosine kinase, JAK2, has been identified in
neonatal and adult rat islets where it is expressed at low abundance,
mainly in the nucleus (131). TrkA, a receptor PTK that acts as a
high-affinity receptor for nerve growth factor, has been localized to
both - and ß-cells of adult islets (132). It has been implicated
in islet development after the observation that K252a, an inhibitor of
Trk receptor PTK activity, inhibited islet morphogenesis (132). Another
neurotrophin receptor PTK, TrkC, has been identified in INS-1
ß-cells, and it shows increased phosphorylation and activation in
response to the receptor ligand neurotrophin-3 (126).
Some PTK substrates (e.g., MAPK, PLC,
phosphatidylinositol-3-kinase, TrkC) have been identified in islets and
ß-cell lines based on their increased tyrosine phosphorylation in
response to inhibition of phosphotyrosine phosphatases or activation of
receptor PTKs (117, 125, 126, 129). In addition, a protein showing
glucose- and insulin-stimulated tyrosine phosphorylation in islets and
in the clonal ß-cell line ßTC3 has been identified immunologically
as the ß-subunit of the insulin receptor (124, 127).
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III. Phosphorylation of Endogenous Proteins in ß-Cells |
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A. Methods for studying protein phosphorylation in ß-cells
Most reported measurements of protein phosphorylation in ß-cells
have used radioisotopic tracer techniques to follow the transfer of
32P or, occasionally, of 35S from radiolabeled
ATP into protein substrates. More recently, immunological methods have
been introduced, using antisera specific for phosphorylated amino acid
residues (phosphotyrosine, phosphoserine/threonine) or for
phosphorylated consensus-site sequences in substrates, and these
methods have proved particularly useful when attempting to identify
substrate proteins (see Table 2).
Radioisotopic studies have used variations of three main technical approaches, each with inherent advantages and disadvantages. The simplest method is to add radiolabeled ATP to a broken cell preparation and to measure the consequent 32P incorporation into endogenous proteins (e.g., Refs. 51, 52, 104). This experimental approach allows the reaction conditions to be precisely controlled, and it permits the detection of rapid (<1 min) phosphorylation events against very low backgrounds of radiolabeled endogenous substrates. However, in studies using broken-cell preparations it is difficult to relate phosphorylation to insulin secretion, and kinases may phosphorylate proteins to which they would normally have no access because of differing intracellular compartmentalization. This may explain why many more kinase substrates have been reported using this experimental approach than by measuring phosphorylation in situ.
These problems can be circumvented by measuring protein phosphorylation
in intact cells, which allows direct comparison of kinase activation
and protein phosphorylation with second messenger generation and
insulin secretion. However, intact ß-cells present several technical
problems: 1) the presence of an intact plasma membrane limits the
extent to which the intracellular pathways can be experimentally
modified; 2) an intact plasma membrane also prevents the supply of
32P as exogenous [32P]ATP, so the
endogenous pool of ATP must be radiolabeled by prolonged incubation of
the intact tissue in the presence of [32P]orthophosphate.
The resultant high level of background phosphorylation can easily
obscure subsequent small stimulus-induced changes; 3) if intracellular
ATP has not achieved isotopic equilibrium, any stimulus-induced changes
in ATP turnover or in [32P]orthophosphate uptake may
alter the specific radioactivity of the intracellular pool of
[32P]ATP.
Islets or cells in which the plasma membranes have been selectively
permeabilized by detergents (133) or by high voltage discharge (134)
offer some of the technical advantages of both homogenates and intact
cells. The permeabilized plasma membranes permit precise control of the
intracellular environment in cells in which the cellular architecture
and compartmentalization are largely unaffected; the radiolabel can be
supplied as [32P]ATP, which allows sensitive detection
of rapid phosphorylation events against very low backgrounds of
radiolabeled proteins; and effects on protein phosphorylation can be
correlated with effects on insulin secretion (134, 135, 136, 137). However,
permeabilized cells are not useful for studying agents that work
entirely or primarily by depolarization of the cell, including the
physiologically important nutrient secretagogues.
B. Endogenous kinase substrates in ß-cells
The presence of kinase activators or insulin secretagogues causes
enhanced phosphorylation of certain proteins in islets or
insulin-secreting cell lines, and these are listed by reported
molecular mass in Table 2. The listings in Table 2 show that a large
number of substrates for regulated kinases have been reported in
ß-cells and there may, on first sight, appear to be little agreement
between different studies. However, most of the reported molecular
masses are derived from migration positions on PAGE, and small
differences in gel composition, running conditions, etc., could produce
small variations in the calculated molecular masses of phosphorylated
proteins, greatly increasing the number of reported substrates and
creating the appearance of disagreement where none actually exists. In
contrast, on some occasions different experimental approaches detect
the same kinase substrates, and these are apparent from the listings in
Table 2 as substrates that have been reported by many different groups
using different ß-cell preparations and technical approaches.
The components of the insulin-secretory process can be arbitrarily classified into four main areas: 1) nutrient transport and metabolism; 2) plasma membrane events; 3) generation of intracellular regulators; 4) secretory granule transport and exocytosis. ß-Cell phosphoproteins have been implicated at each of these different stages.
1. Nutrient transport and metabolism. Glucose transport across the ß-cell plasma membrane is not considered to be rate limiting for insulin secretion (138), although it has been demonstrated that PKA activation by forskolin results in rapid serine/threonine phosphorylation of the ß-cell glucose transporter, GLUT2 (139). Once inside the ß-cell, nutrient secretagogues must be metabolized to stimulate insulin secretion (140), and the phosphorylation state of metabolic enzymes often regulates their activity. A 57-kDa substrate for CaMK was reported to react with antipyruvate kinase antibodies (141), but this identification has not been confirmed, and there is little evidence in ß-cells that protein kinases control secretion by regulating metabolic functions.
2. Plasma membrane events. Pancreatic ß-cells are excitable cells that depend upon depolarization of the plasma membrane as a means of responding to some external stimuli. There is some evidence that the phosphorylation state of ion channels in ß-cell plasma membranes may influence their function (142). For example, it was suggested on the basis of electrophysiological evidence that in RINm5F ß-cells the KATP channels are targets for PKC (143) or unspecified protein kinases (144), but the relevance of this to physiological secretory responses is unclear since PKC activation induced insulin release from normal mouse ß-cells without modifying the membrane potential (145). Independent cloning studies have now indicated that there are at least three subfamilies of the inward rectifier K+ channel family expressed in ß-cells, designated uKATP-1, KATP-2, and Kir6.2 (146, 147, 148), and that the sulfonylurea receptors are members of the ATP-binding cassette superfamily (149). The sulfonylurea receptor does not have intrinsic channel activity but confers sulfonylurea sensitivity on a variety of inward rectifier K+ channels (150, 151). Both the KATP channels and the sulfonylurea receptors have several potential phosphorylation sites for PKC and PKA (146, 147, 149), suggesting that their activities may be modulated by these kinases. Future studies using channels and sulfonylurea receptors mutated at potential phosphorylation sites will provide insights into the physiological relevance of channel phosphorylation in the regulation of ß-cell electrical excitability.
Depolarization of the ß-cell causes the opening of voltage-dependent
Ca2+ channels (VDCC) and the 1- and
ß3-subunits of the ß-cell L-type Ca2+
channel have been cloned; the 1-subunit acts as a
voltage sensor and forms the ion-conducting pore, and the
ß3-subunit modulates channel activity (152). A
ß-subunit cloned from brain, and also detected in RINm5F and ßTC3
cells, encodes numerous consensus phosphorylation sites (153) whereas
the ß-cell 1-subunit cloned from human ß-cells
contains 11 potential phosphorylation sites for PKA, 9 for PKG, and 1
for PKC (154, 155). There is much early experimental evidence, mainly
from studies of insulin secretion and Ca2+ influx/efflux,
that cAMP can modulate Ca2+ handling by ß-cells (156),
although the significance of this to the secretory response is still
not clear. More recently, the activation of PKA has been shown directly
to increase the phosphorylation of the 1 VDCC subunit in
ßTC3 cells, suggesting a role for PKA in the regulation of
Ca2+ influx in these insulinoma cells (157). It has also
been suggested that PKC may regulate Ca2+ influx into
ß-cells by changing the phosphorylation state of the L-type VDCC
(158), although there has, as yet, been no direct demonstration of
PKC-mediated phosphorylation of ß-cell VDCC subunit(s). As for the
KATP channel, it is clear that future measurements of the
functional consequences of modifying VDCC subunits will delineate the
importance of these phosphorylation events in the initiation and
maintenance of ß-cell secretory responses.
Although it seems likely that ß-cell ion channels are targets for phosphorylation by regulated kinases, such effects alone cannot account for the stimulatory effects of second messengers on insulin secretion since cAMP and activators of PKC can stimulate insulin secretion from permeabilized islets or from single voltage-clamped ß-cells independently of ion fluxes (29, 159, 160, 161, 162, 163, 164).
3. Generation of intracellular regulators. The ß-cell signal
transduction systems that generate intracellular regulators may
themselves be modified by phosphorylation. Receptor-mediated,
nonnutrient secretagogues often influence intracellular events through
G proteins, which are known to be substrates for kinases in other
tissues (165, 166, 167), and there is some indirect evidence that insulin
secretagogues stimulate the phosphorylation of ß-cell G proteins. For
example, the phosphorylation of Gi by PKC may modulate
ß-cell AC activity (168, 169, 170), and a PKC substrate of the appropriate
molecular mass (40 kDa) for the -subunit of Gi has
been reported in several studies (Table 2). The enzymes regulated by G
proteins may also be targets for protein kinases. In RINm5F cells the
-isoform of PLC is tyrosine phosphorylated (129), while in neonatal
rat islets cPLA2 is phosphorylated by PKC (123).
In addition to phosphorylating other protein substrates, it is known
that CaMK, PKC, and PKA are themselves kinase substrates, and p42/44
MAP kinases require threonine and tyrosine phosphorylation for
activation (Section II.D). The subunits of CaMK II
autophosphorylate in the presence of Ca2+ and CaM and thus
become much less Ca2+ dependent (41). It seems probable
that the ß-cell proteins of molecular masses 53–55 kDa, which have
been reported by many groups to be substrates for
Ca2+-dependent phosphorylation (Table 2), are
autophosphorylated CaMK II subunits (49, 58). The RII-regulatory
subunit of type II PKA can be phosphorylated by the catalytic subunit
(44), and it has been suggested that a 54-kDa islet protein, which is a
substrate for PKA and which binds azido-cAMP, may be an
autophosphorylated regulatory subunit of PKA (74). Similarly, it has
been suggested that the 80-kDa islet protein substrate for PKC is
autophosphorylated PKC (171), although it is more likely to be the
myristoylated alanine-rich C kinase substrate (MARCKS) protein, which
is a filamentous actin cross-linking protein of approximately 80-kDa
molecular mass whose function is modified by PKC-mediated
phosphorylation (172). Enhanced phosphorylation of MARCKS in response
to PKC-activating phorbol esters, to cholinergic agonists, and to
glucose has been reported in rat and mouse islets (103, 107, 173).
The regulation of effector systems by phosphorylation may not be responsible for the initiation of insulin-secretory responses but may play an important role in coordinating responses to different stimuli and in determining the rate and extent of the response. There is some evidence for cross-talk between signaling systems in ß-cells, and this has been reviewed elsewhere (174). At present, little of the experimental evidence for cross-talk in ß-cells is at the level of kinase activation or protein phosphorylation, and so this interesting area is beyond the scope of the present review. Whatever the importance of phosphorylation in modifying the generation of intracellular regulators, the effects of those regulators on secretion from permeabilized and patch clamped cells (159, 160, 161, 162, 163, 164) implies that there exist downstream from the generation of the regulators kinase substrates whose phosphorylation state regulates the secretory response.
4. Secretory granule transport and exocytosis. Cytoskeletal
elements such as actin, myosin, and tubulin are involved in the
movement of insulin-secretory granules within the ß-cell (175), and
thus offer potential sites at which kinases may regulate secretory
responses. There is some evidence implicating cytoskeletal proteins in
regulated phosphorylation events reported in ß-cells. For example,
the 20-kDa protein substrate detected in several studies (Table 2) is
almost certainly myosin light chain, which acts primarily as a
substrate for islet MLCK (48, 50). The 57-kDa and 54-kDa islet
substrates for Ca2+-dependent phosphorylation were
identified as the - and ß-subunits of tubulin on the basis of
their electrophoretic mobilities and immunoreactivity with antitubulin
antibody (54), but this identification was disputed on the basis of the
isoelectric point of the 57-kDa protein (74). The 80-kDa MARCKS protein
is an actin-cross-linking protein that shows stimulus-dependent
increases in phosphorylation in rat and mouse islets (103, 107, 173).
The exocytotic release of secretory granule contents requires the
docking of secretory granules at the site of exocytosis, a priming
process that renders the granule competent for exocytosis, followed by
fusion of granule membranes with the plasma membrane. As detailed in
Table 2, a number of unidentified kinase substrates have been localized
to ß-cell secretory granules or membrane fractions, and some of these
may be involved in the exocytotic docking and/or fusion processes.
Advances in our understanding of the general mechanisms of exocytosis
have been reviewed recently (e.g., Refs. 176, 177), and a
full discussion of this topic in ß-cells has been the subject of a
comprehensive review (15). In brief, vesicles are directed to the site
of exocytosis by interactions between receptors located on the
secretory vesicle membrane (v-SNAREs) or on the target membrane
(t-SNAREs), a process that is regulated by the low molecular mass G
proteins of the Rab family. Membrane fusion events are thought to be
triggered by interaction of N-ethylmaleimide-sensitive
fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs)
with the SNARE complex in an ATP-hydrolyzing reaction. Recent studies
have shown that ß-cells express NSF and its attachment protein
SNAP (178), Rab3 proteins (179), the vesicle-associated SNARE
proteins, VAMP and cellubrevin (180, 181), and the plasma-membrane
associated target SNARE proteins, synaptosome-associated protein-25
(SNAP-25) and syntaxin 1–4 (181, 182, 183, 184). A recent in vitro
study using recombinant proteins demonstrated that VAMP and SNAP are
phosphorylated by CaMK II and PKA, respectively (185), although there
is as yet no evidence that any of these proteins are phosphorylated in
ß-cells. A recent report suggests that SNAP-25 is tyrosine
phosphorylated in RIN 1046–38 ß-cells in response to a combination
of glucose and glucagon-like peptide-1 (GLP-1), although the kinase
responsible has not been identified (186). Other proteins that have
been implicated in exocytosis have been identified as phosphoproteins
in ß-cells. For example, annexin-I, a protein implicated in membrane
fusion events (187), is present in insulin-secretory granules and is
phosphorylated in a glucose-dependent manner (188); and an 84-kDa
secretory granule-associated synapsin protein is phosphorylated by CaMK
II in glucose-stimulated MIN6 ß-cells (58).
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IV. Protein Kinases and the Regulation of Insulin Secretion |
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TopAbstractI. IntroductionII. Protein Kinases in...III. Phosphorylation of...IV. Protein Kinases and...V. Protein Phosphatases and...VI. Protein Kinase-Independent...VII. Summary and Future...References |
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The most commonly used protein serine/threonine kinase inhibitors are those that inhibit phosphorylation by interacting with the ATP-binding site of the enzyme, but inhibitors of this type tend to be poorly selective since the ATP-binding sites of the common regulated serine/threonine protein kinases are very similar (reviewed in Refs. 35, 37, 189). More recently developed ATP-binding site inhibitors appear to show a higher degree of selectivity (190, 191), although it is uncertain whether this approach will permit specific inhibition of individual kinase isoforms. Inhibitors that act by interfering with the binding of the intracellular activators (e.g., Ca2+/CaM, cAMP, DAG) offer potentially more selectivity between classes of regulated kinases (192, 193, 194), although these inhibitors may not differentiate between kinase isoforms, and they may also inhibit binding of the activators to response elements other than protein kinases. A greater degree of selectivity can be obtained using consensus site-derived pseudosubstrate peptide sequences that bind to and inhibit the substrate site of the kinases (195), but the unmodified peptide sequences are poorly membrane permeant, which has largely limited their use to single-cell microinjection or patch-clamp preparations (164, 196) and to populations of permeabilized cells (197, 198). Chemical modification of the peptides can make them more membrane permeant (199), and this approach has been applied to peptide inhibitors of PKC (200) and PKA (201) in ß-cells.
The problems of inhibitor specificity and delivery can be circumvented
by generating ß-cells deficient in the enzyme of interest. One useful
pharmacological approach, that of down-regulation of the DAG-sensitive
conventional and novel isoforms of PKC by prolonged treatment with
phorbol esters, has been widely applied to study the roles of PKC in
ß-cells, as described in Section IV.C. Transfection of
ß-cells with antisense nucleotide sequences to prevent the expression
of particular protein kinases is feasible (202, 203) but, as with the
inhibitory peptides, the major technical problem lies with introducing
sufficient amounts of the antisense sequence into populations of cells
in a reproducible manner. Stable antisense transfects to prevent kinase
expression have not yet been reported for ß-cells, but this may offer
an effective means of selectively depleting cells of kinase isoforms.
Conversely, stable transfection of RINm5F cells with a viral vector
coding for the -isoform of CaMK II has been reported (204), and this
overexpression approach may provide useful information complementary to
inhibitor studies. Transgenic animal models are now becoming more
widely available, and several protein kinase knockout animals have been
reported (205, 206, 207), although information is not yet available about
ß-cell function in these models. This technical approach has also
been used in conjunction with a ß-cell-specific promoter to
overexpress CaM selectively in ß-cells, which results in severely
diabetic neonates with reduced ß-cell number and abnormal ß-cell
morphology (208). These defects were later attributed to
Ca2+ buffering by the overexpressed CaM, rather than to the
activation of Ca2+/CaM-dependent effectors within the
ß-cells (209). A similar approach has been used to modify the
cAMP/PKA signaling pathway in ß-cells either by overexpressing
cholera toxin, which irreversibly activates Gs (210), or by
overexpressing a constitutively active -subunit of Gs
(211).
Given the uncertainties inherent in many of the individual approaches used to study kinase function in ß-cells, the sequential application of disparate methods seems more likely to produce an accurate evaluation of the importance of a particular kinase/isoform in the generation and/or maintenance of secretory responses to a specific stimulus. Accordingly, we suggest that, wherever possible, the following criteria should be satisfied to indicate the involvement of a particular protein kinase/isoform in physiological insulin-secretory responses.
1. Nutrient and/or nonnutrient secretagogues should generate activators of the kinase(s) at the appropriate concentrations, time, and intracellular locations.
2. Physiologically relevant concentrations of the secretagogue(s) should induce kinase activation and phosphorylation of endogenous protein substrates.
3. Experimentally induced elevations in intracellular concentrations of the kinase activators should stimulate protein phosphorylation and thus modify insulin secretion.
4. Blocking protein phosphorylation using structurally and, where possible, mechanistically dissimilar kinase inhibitors should inhibit phosphorylation and secretory responses to physiological secretagogues.
5. ß-Cells deficient in the protein kinase/isoform should exhibit appropriately diminished secretory responses to physiological agents.
Using this approach it is possible to define, to some extent, the involvement of protein kinases in the regulation of insulin secretion even where the identities and functions of the kinase substrates are as yet unknown.
A. Ca2+/calmodulin-dependent protein kinases
Changes in intracellular Ca2+ concentrations play a
vital role in the regulation of insulin secretion by both nutrient and
nonnutrient secretagogues, and the complexities of stimulus-induced
changes in ß-cell Ca2+ have been covered in many
excellent reviews (e.g., Refs. 6, 7, 8, 9, 16, 212). Stated
simply, nutrient secretagogues elevate cytosolic Ca2+
primarily through an influx of extracellular Ca2+, while
some receptor-operated nonnutrient secretagogues generate
IP3, which increases cytosolic Ca2+ by
mobilizing intracellular stores of Ca2+. Sulfonylureas,
cationic amino acids (e.g., arginine) and K+
induce Ca2+ entry through VDCC by depolarizing the ß-cell
plasma membrane, whereas Ca2+ ionophores such as A23187
facilitate the transport of Ca2+ across membranes.
Experiments using permeabilized islets have demonstrated that
increasing the intracellular concentration of Ca2+ (from
50 nM to 1–10 µM) is alone a sufficient
stimulus to induce an ATP-dependent stimulation of insulin secretion
(133, 160). More recently, measurements of exocytosis from single
ß-cells by monitoring changes in membrane capacitance have confirmed
that elevated Ca2+ is a sufficient trigger for exocytosis
(164).
In common with other secretory cells, pancreatic ß-cells express a variety of Ca2+-sensitive proteins that may be involved in sensing and responding to changes in cytosolic Ca2+, and CaMK II is one likely candidate for transducing Ca2+-mobilizing signals into a secretory response. Thus, the Ca2+/CaM-dependent phosphorylation of 54- to 57-kDa proteins in rat islet microsomal fractions (54) was also detected in permeabilized islets in situ and was accompanied by Ca2+-induced insulin secretion (133, 134). A correlation between phosphorylation and insulin secretion was also observed in 32P-labeled intact islets in which glucose stimulated both insulin release and phosphorylation of the 54-kDa protein (54). In more recent experiments, measurements of CaMK II autophosphorylation, and subsequent Ca2+-independence, have been used as a reflection of CaMK activation in intact islets. Glucose or depolarizing concentrations of K+ caused rapid, concentration-dependent increases in CaMK activity as a result of Ca2+ entry through VDCC (213, 214), and a similar activation was caused through the mobilization of intracellular Ca2+ by the muscarinic agonist CCh (214). The glucose concentration dependence of CaMK II activation was closely correlated to the stimulation of insulin secretion, suggesting that the two events may be linked, and CaMK II activation preceded the initiation of insulin secretion, as would be required if CaMK II was playing a causal role in the secretory response (213). The increase in autophosphorylated autonomous CaMK II activity in response to CCh was rapid (214), in accordance with the reported rapid effects of muscarinic agonists on ß-cell Ca2+ (158, 215, 216, 217), but it was transient and returned to basal levels within 1–2 min (214). In static incubations, CaMK II activation by glucose was also relatively transient, returning to unstimulated levels within 20 min (213), although elevations in ß-cell cytosolic Ca2+ and insulin secretion are maintained for the duration of a glucose stimulus (103, 218, 219). The observations in intact islets in static incubations parallel observations in perifused permeabilized islets and ß-cells that insulin-secretory responses to elevations in Ca2+ are transient (136, 220, 221), with the loss of secretory responsiveness to Ca2+ being accompanied by a reduction in CaMK activity (136), and suggest that the activation of CaMK II cannot alone account for the maintained secretory responses to nutrient secretagogues. In contrast to the data obtained in static incubations of intact islets and perifused permeabilized islets, there has been a recent report that CaMK II activation is closely correlated with glucose-stimulated insulin secretion for up to 30 min when measured in perifusion experiments (222), suggesting a relationship between CaMK II activity and maintained insulin secretion. More studies examining the time course of CaMK II activation are required to resolve whether CaMK is involved not only in the initiation of nutrient-stimulated insulin secretion, but also in its maintenance.
Studies using inhibitors of CaMK II generally support a role in the regulation of insulin secretion in response to Ca2+-mobilizing stimuli. Early investigations of the involvement of CaMKs in glucose-stimulated insulin secretion made use of the CaM antagonist, TFP, which inhibited glucose-stimulated insulin secretion without affecting glucose oxidation (223). In a Syrian hamster insulinoma both Ca2+/CaM-stimulated phosphorylation of 60-kDa and 98-kDa proteins and K+-stimulated insulin secretion were inhibited by TFP (47, 224). Similarly, TFP abolished phosphorylation of a 55-kDa CaMK substrate in rat islets and inhibited insulin secretion stimulated by glucose, leucine, and glibenclamide (225). However, TFP is not a direct inhibitor of CaMK, but is a CaM antagonist and has been shown to exert inhibitory effects on other CaM-dependent enzymes, and on other protein kinases including PKC (226). An alternative CaMK inhibitor, dehydrouramil, inhibited CaMK activity in islet extracts with little effect on PKA or PKC activities, and dehydrouramil inhibited glucose-induced insulin secretion from intact rat islets (227). More recently, at least four separate studies have demonstrated that the novel CaMK II inhibitor, KN-62, inhibits nutrient-induced insulin secretion (61, 228, 229, 230). KN-62 is a competitive inhibitor at the CaM-binding site and had no effect on RINm5F cell PKC activity or on MLCK activity (229). However, in HIT cells the inhibitory effects of KN-62 on nutrient- and K+-stimulated insulin secretion were coupled to an inhibition of Ca2+ entry, and it did not inhibit insulin release stimulated by Ca2+ in permeabilized HIT cells (228). The inhibitory effect of KN-62 on Ca2+ current has also been observed in single mouse ß-cells, but in this model KN-62 also caused a decrease in depolarization-induced membrane capacitance, indicative of an inhibition of exocytosis (164). Peptides directed against regulatory domains of CaMK II offer an alternative means of inhibiting enzyme activity, and synthetic sequences corresponding to amino acids 281–309 and 290–309 of the rat brain 50-kDa CaMK II subunit are potent inhibitors of islet CaMK II activity in vitro (our unpublished data). Administration of peptide 290–309 to mouse ß-cells via a patch pipette resulted in a 60% reduction in depolarization-induced increase in membrane capacitance, without any effect on the Ca2+ current (164), suggesting that the activation of CaMK II is required for full exocytotic responses after the depolarization-induced elevations in Ca2+.
Most investigations of CaMKs in ß-cells have focused on the
multifunctional CaMK II rather than on CaMK III or MLCK. There is no
evidence that CAMK III is involved in the regulation of insulin
secretion, although the identification of elongation factor 2 as the
major CaMK III substrate in islets suggests a role in the regulation of
ß-cell protein synthesis (55). In contrast, there is some
circumstantial evidence that MLCK may be involved in the secretory
process, although the strength of the evidence depends entirely on the
selectivity of the inhibitors employed. Thus, an MLCK inhibitor, ML-9,
inhibited phosphorylation of myosin light chains by islet extracts and
also caused a small (30%) inhibition of glucose-stimulated insulin
secretion (230); glucose-induced insulin secretion from MIN6 ß-cells
was totally inhibited by the fungal metabolite, wortmannin, which is an
inhibitor of MLCK at the high concentrations used in this study (231);
and there is one report that antibodies against MLCK inhibit insulin
release from permeabilized islets (232).
On balance, the available experimental evidence suggests that CaMK II
is involved in mediating ß-cell-secretory responses to
Ca2+-mobilizing stimuli, and data from a number of groups
fulfil criteria 1–4: the effects of insulin secretagogues on
increased intracellular Ca2+ in ß-cells correlate
with increased CaMK II activity and the phosphorylation of endogenous
CaMK II substrates; experimental elevations in ß-cell cytosolic
Ca2+ stimulate phosphorylation and insulin secretion; and a
number of CaMK inhibitors inhibit insulin secretion. CaMK II may
therefore be responsible for the initiation of an insulin-secretory
response to nutrient secretagogues, and for enhancing nutrient-induced
responses by IP3-generating agonists, as depicted in Fig. 1. However, the time courses of CaMK II
activity and of Ca2+-induced insulin secretion suggest that
the activation of CaMK II is alone unlikely to produce a physiological
pattern of prolonged insulin secretion. If this is the case, the
maintained elevations in cytosolic Ca2+ in response to
nutrients may subserve another function, perhaps by activating a
Ca2+-response element other than CaMK II that is required
for the maintenance of a prolonged secretory response.
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In contrast, there have been numerous reports over the past two decades
that experimentally induced elevations in ß-cell cAMP can enhance
insulin secretion. The reported effects are remarkably consistent
whether ß-cell cAMP was elevated by activating AC using forskolin or
cholera toxin through inhibition of cAMP degradation by
phosphodiesterases (reviewed in Refs. 7, 8, 12); by introducing
cAMP in permeabilized ß-cells (159, 161, 237); or by using
membrane-permeant analogs of cAMP in intact ß-cells (238, 239).
Studies using transgenic mice showing ß-cell-specific expression of
the AC-activating A1 subunit of cholera toxin found surprisingly little
change in ß-cell morphology, cAMP content, or insulin-secretory
responses to glucose (210), suggesting the existence of a
counterregulatory mechanism to prevent unregulated elevations in
ß-cell cAMP. Similarly, overexpression of the -subunit of the
AC-linked Gs did not affect cAMP content or secretory
responses of islets or perfused pancreas to glucose alone, but the
stimulatory effects of the phosphodiesterase inhibitor
isobutylmethylxanthine (IBMX) were enhanced (211). These studies
suggest that ß-cells have an unexpected capacity to adapt their
physiological function in the event of up-regulation of AC activity,
which may complicate interpretation of experiments in which there are
maintained, rather than short-term, alterations in the status of key
regulatory molecules.
Until recently, studies of the physiological functions of PKA were
hampered by the lack of specific, membrane-permeant inhibitors of the
enzyme. In an early report, 2-deoxyadenosine, an inhibitor of islet
AC activity (240), blocked IBMX-induced phosphorylation of a 15-kDa
islet protein, with a concomitant reduction in IBMX-induced insulin
secretion (233), but the effects of this inhibitor on secretory
responses to physiologically relevant nutrients or nonnutrients were
not investigated. More recently, membrane-permeant competitive
antagonists of cAMP have been developed and shown to inhibit
PKA-mediated events in islets (137), single ß-cells (29, 163, 241),
and -cells (242). Rp-cAMPS has been used to block protein
phosphorylation (137) and insulin-secretory responses induced by cAMP
and forskolin (137, 163, 241) to demonstrate that these events are PKA
dependent. More recently, GIP-induced exocytosis in patch-clamped
ß-cells was suppressed by Rp-8-Br-cAMPS (29), suggesting that GIP and
other AC-linked, nonnutrient secretagogues enhance insulin secretion
primarily through the activation of PKA (Figure 2).
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Although PKA activation is normally not sufficient to initiate insulin secretion, it remains a possibility that PKA activation is required for secretory responses to nutrients. There have been a number of reports that poor or absent ß-cell responses to glucose can be remedied by agonist-induced or pharmacological elevations in intracellular cAMP in isolated patch-clamped ß-cells (241), in purified populations of ß-cells (215, 243, 244), and in freshly isolated islets (211, 245), implying that the ß-cell content of cAMP, and thus the activation state of PKA, is a vital component in nutrient-induced insulin secretion. In contrast to this, glucose-induced insulin secretion from freshly isolated islets was not significantly inhibited by Rp-cAMPS (137) or by a membrane-permeant, myristoylated peptide inhibitor of PKA (201), although both inhibitors were shown to inhibit phosphorylation and secretory responses to cAMP and forskolin. Experimental models in which the expression of ß-cell PKA is suppressed are not yet available. Such models could determine decisively whether the activation of PKA is obligatory for nutrient-induced insulin secretion and the extent to which nonnutrients modify ß-cell responses by PKA-dependent protein phosphorylation.
Islets of Langerhans also express cyclic GMP-dependent protein kinase
(PKG) activity (78), and a role for cyclic GMP in the stimulation of
insulin secretion has been proposed (246). Glucose produces small
increases in islet cyclic GMP content, although the time course of the
onset of this effect varies from 2–3 min (247) to at least 30 min
(248), whereas the secretory response to glucose is maximal within
approximately 2–3 min, perhaps suggesting that the two events are not
causally related. There is also no clear consensus on the effects of
experimental elevations in ß-cell cyclic GMP, which are reported to
be stimulatory (246, 249), inhibitory (250), or without effect (248).
Some cytokines, such as interleukin-1ß and tumor necrosis factor-,
produce nitric oxide-mediated increases in islet cyclic GMP content
(251, 252) and also inhibit glucose-induced insulin secretion, but the
effects on insulin secretion can be dissociated from cyclic GMP
accumulation (253). The activation of PKG therefore seems unlikely to
play a major role in the regulation of ß-cell secretory responses to
metabolic stimuli or to receptor-operated agonists. However, a recent
report has implicated cyclic GMP, and by implication PKG, in the
induction of apoptotic cell death in ß-cells (254). It remains an
intriguing and therapeutically important possibility that manipulation
of PKG activity may influence ß-cell survival in the face of
apoptotic stimuli, and this is surely an area worthy of further study.
C. Ca2+/phospholipid-dependent protein kinases
The role(s) played by members of the PKC family of isoenzymes in
the regulation of insulin secretion has been the subject of numerous
studies over the past decade or so, many of which have produced
conflicting data. This area of ß-cell research has been reviewed
extensively elsewhere (e.g., Refs. 255, 256, 257, 258), and readers
are referred to one or more of these reviews for detailed comment,
while we will limit ourselves to a broad consideration of the criteria
outlined in Section IV.
There is no doubt that physiological regulators of insulin secretion
can generate (potential) activators of PKC within ß-cells. The major
physiological regulators of ß-cell PKC activity are likely to be
DAGs, which activate isoforms of PKC containing two C1 cysteine-rich
repeats (all isoforms except , /, and µ). A variety of
receptor-operated nonnutrient secretagogues, including CCh, CCK, and
AVP, activate ß-cell PLC via Gq leading to the hydrolysis
of phosphatidylinositol bisphosphate (PIP2) and the
generation of DAG (22, 25, 258). Nutrient secretagogues also increase
ß-cell DAG content, but by two independent methods: by enhancing
de novo synthesis of DAG (259) and by a
Ca2+-dependent activation of PLC (22). It is important to
note that PLC activation appears to contribute little to the
glucose-induced increase in DAG mass within islets, which is
predominantly enriched in palmitic acid, indicative of synthesis
de novo (259), rather than stearoyl-arachidonyl
configuration indicative of hydrolysis from PIP2. It is
also worth noting that PLC-generated stearoyl-arachidonyl DAG is a much
more potent activator of PKC in vitro than is the species of
DAG synthesized de novo (260), which may be an important
clue as to the roles of PKC in nutrient and receptor-operated secretory
responses. The DAG-sensitive isoforms of PKC can also be activated by
long chain unsaturated fatty acids such as AA, which have been shown to
activate islet PKC in vitro (85, 86) and which may be
important regulators of insulin secretion (261). AA can also be
converted to DAG by DAG lipase, providing an alternative source of DAG
for PKC activation. Both nutrient and nonnutrient secretagogues
activate ß-cell PLA2, thus stimulating AA production by
the hydrolysis of phosphatidylcholine (26, 262, 263).
There is also no doubt that the activation of DAG-sensitive isoforms of
PKC within ß-cells stimulates insulin secretion. It is well
documented that a variety of DAG analogs, including the tumor-promoting
phorbol esters, cause prolonged insulin-secretory responses in isolated
islets and ß-cell suspensions (reviewed in Ref. 257). The onset of
the insulin-secretory response of islets to 4ß-phorbol myristate
acetate (PMA) closely parallels the time course of the second phase of
insulin secretion in response to glucose, and this has been interpreted
as being indicative of a role for PKC in mediating this phase of
glucose-stimulated insulin release (264). The effects of PKC activators
on insulin secretion are accompanied by effects on protein
phosphorylation, and several PKC substrates have been identified in
both islets and insulin-secreting cell lines stimulated with phorbol
esters (see Table 2). In some instances, glucose and/or CCh have
similar effects to those seen after the direct activation of PKC,
perhaps suggesting common activation of the same kinase (Ref. 257 and
references therein). The most convincing evidence of PKC activation by
a physiologically relevant insulin secretagogue has been the
observation, using two-dimensional SDS-PAGE, that CCh stimulates the
phosphorylation of the PKC substrate MARCKS (103, 173). An effect of
nutrients on MARCKS phosphorylation is less convincing. In one study,
glucose clearly stimulated 32P incorporation into MARCKS,
to the same extent as that caused by PMA (107), but other studies have
reported negligible effects of glucose on MARCKS phosphorylation,
certainly much less than those caused by CCh or PMA (103, 173).
As discussed in Section II.C, the activation of
DAG-sensitive PKC isoforms is accompanied by translocation of the
enzyme from the cytosol to a membrane compartment (93), and this
physical redistribution of PKC has been widely used to assess PKC
activation in response to insulin secretagogues, either by measuring
the activity of DAG-sensitive isoforms of the enzyme in cytosolic and
membrane fractions, or by immunodetection of the distribution of
particular PKC isoforms. Both PMA and CCh stimulate PKC redistribution
between subcellular fractions, whether measured by phosphorylation of
an exogenous substrate or by quantitative immunoblotting (88, 89, 94, 260, 265, 266). This experimental approach therefore supports a role
for PKC activation in secretory responses to PLC-coupled nonnutrient
secretagogues. The effects of nutrients are not so clear. Although
there is one report of a glucose-induced increase in
membrane-associated PKC activity (90), most studies agree that
nutrients such as glucose and glyceraldehyde do not stimulate the
translocation of PKC enzyme activity in islets or ß-cells (88, 89, 96, 260, 267). In contrast, immunoblotting and immunocytochemical
approaches have shown nutrient-induced translocation of the -
(94, 95, 107, 112) and - (112) isoforms of PKC. The reasons for
these discrepancies between experimental approaches are not clear, but
the existence of such conflicting observations raises questions about
using PKC translocation as the sole measure of its involvement in
secretory responses to nutrients.
A range of inhibitors have been employed in attempts to define the role
of PKC in ß-cells, but many of these studies have merely added to the
confusion about whether or not PKC is required for a secretory response
to nutrients. A variety of kinase inhibitors, including H-7,
clomiphene, polymyxin B, and staurosporine, inhibited PKC activity and
insulin secretion in response to glucose (89, 268, 269, 270, 271), to cholinergic
agonists (173, 268, 272), and to CCK (273), suggesting that PKC
activation is required for a full secretory response to nutrients and
PLC-activating nonnutrients. However, it is difficult to draw firm
conclusions from these observations since all of these compounds are
relatively nonselective kinase inhibitors (274, 275). For example,
staurosporine has been widely used as a PKC inhibitor in ß-cells, but
the original description of staurosporine presented it as an equipotent
inhibitor of PKA and PKC (276), and it is a potent inhibitor of islet
PKA and CaMK activities (271). More recently developed PKC inhibitors
show improved selectivity for PKC and some degree of selectivity for
PKC isoforms. The staurosporine derivative Ro 31–8220 inhibited all
PKC isoforms tested (277) and partially inhibited glucose-stimulated
insulin release (278, 279). In contrast, glucose-induced insulin
secretion from rat islets was not inhibited by a myristoylated peptide
inhibitor of the - and ß-isoforms of PKC (200), or by the
indolocarbazole Gö 6976 (279), which shows selectivity for the
conventional isoforms of PKC [, ß, (191)]. These
observations might suggest that nutrient-induced insulin secretion
requires the activation of a PKC isoform(s) other than the
Ca2+/DAG-sensitive conventional isoforms, although maximal
inhibition of PKC by Ro 31–8220 reduced glucose-induced secretory
responses by only 50%, implying the existence of another,
PKC-independent pathway(s).
Studies of the physiological functions of PKC have been greatly
assisted by the phenomenon of down-regulation of PKC activity by
prolonged exposure to PKC-activating phorbol esters, as described in
Section II.C, which produces cells deficient in some
DAG-sensitive isoforms. Measurements of enzyme activity show marked
reductions (>90%) in DAG-dependent PKC activity in islets and
ß-cells after overnight exposure to phorbol esters (e.g.,
Refs. 91, 102, 104, 273, 280), which is accompanied by the loss of
immunoreactive -, -, and -isoforms of PKC but not,
surprisingly, of the DAG-sensitive ßII isoform (111). The total loss
of secretory responsiveness of PKC down-regulated ß-cells to DAG
analogs implies that -, -, and -isoforms may be involved in
regulating secretion, but the DAG-sensitive ßII isoform that persists
after PMA treatment plays no important role in secretion in response to
these agents. As would be expected, expression of the atypical PKC
isoforms (, /, and µ) was unaffected in ß-cells after
24 h exposure to the phorbol ester (111).
PKC down-regulated ß-cells have been used extensively to investigate the role played by phorbol ester-sensitive isoforms of PKC in the potentiation of insulin secretion by hormones and neurotransmitters, and these studies have, in general, shown good agreement among different laboratories. PKC depletion of islets or HIT-T15 cells resulted in reduced secretory responses to cholinergic agonists (102, 216, 281, 282) to CCK (273, 283), and to AVP (216). In contrast, PKC depletion enhanced secretory responses of RINm5F cells to CCh and AVP (92), which may reflect the expression in this cell line of different PKC isoforms to those expressed in normal ß-cells (106). This approach has also been applied to studies of nutrient-induced insulin secretion, and there is, as always, some disagreement in the literature. Several different groups have reported that PKC-depleted islets mount similar secretory responses to control islets treated with inactive phorbol esters, whether challenged with glucose (e.g., Refs. 101, 103, 135, 169, 281) or glyceraldehyde (267). In contrast, down-regulation of PKC has been reported to cause a marked reduction in the second phase of glucose-induced secretion from perifused mouse islets (104), although this effect was not observed in similar studies using mouse (103) or rat islets (281) in which neither phase of the secretory response to nutrients was significantly inhibited by PKC depletion. Confusing effects of nutrients have been reported in studies using the glucose-insensitive RINm5F cell line, in which glyceraldehyde-stimulated insulin secretion has been reported to be either decreased (91) or increased (92) in PKC-depleted cells.
What model could encompass the confusing and sometimes conflicting
observations using PKC activators, inhibitors, and PKC down-regulation?
The stimulus-response coupling of nonnutrients through PKC to a
secretory response seems to be fairly well established. Thus, there is
general agreement using all of these experimental approaches that PKC
activation is required for a full response to receptor-mediated
secretagogues that are linked to PLC: such stimuli generate DAG,
activate and translocate PKC, and induce PKC-dependent phosphorylation
of endogenous substrates; their effects are blocked by a range of PKC
inhibitors; and down-regulation studies have identified one or more of
the -, -, and -isoforms as the proteins mediating these
responses to nonnutrients in ß-cells. In contrast, our view is that
these isoforms of PKC do not play an important role in the initiation
of secretory responses to nutrients. Thus, the evidence that nutrients
activate the DAG-sensitive isoforms of PKC is not entirely convincing,
and down-regulation studies do not support an obligatory role for
DAG-sensitive isoforms in the initiation of nutrient-induced secretion.
However, this conclusion does not preclude a role for PKC in
nutrient-induced insulin secretion: PKC down-regulation experiments
tell us nothing about the DAG-insensitive -, /-, and µ-
isoforms that are expressed in ß-cells (108, 111, 113, 114), and we
cannot ignore the evidence of inhibition of nutrient-induced secretion
by PKC inhibitors, particularly the more recently developed
PKC-selective compounds. If a DAG-insensitive atypical PKC isoform(s)
is involved in the response to nutrients, the reported inhibitory
effects of Ro 31–8220 on glucose-induced insulin secretion could be
explained by its effects on all PKC isoforms — conventional, novel and
atypical — whereas the observed lack of effect of Gö 6976
reflects its selectivity for inhibition of conventional isoforms.
We therefore propose a model (Figure 3)
in which nutrients stimulate insulin secretion partly through the
activation of an atypical isoform(s) of PKC (, /, and/or µ),
whereas the potentiation of nutrient-induced secretion by
PLC-activating nonnutrients is transduced through the activation of one
or more of the -, -, and/or -isoforms. The intracellular
mechanisms by which nutrients could activate atypical PKC isoforms in
islets are unknown, but one possible candidate is phosphatidylinositol
3,4,5-trisphosphate, which has been shown to activate PKC in
vitro (284) and to be generated in islets by the activation of
phosphatidylinositol 3-kinase in response to nutrients (285).
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D. Mitogen-activated protein kinases
MAPK cascades have been implicated in signal transduction pathways
in many mammalian cells, including specialized secretory cells that are
not mitogenically responsive, such as adrenal chromaffin cells (286).
To date, little information is available on the regulation and roles of
the MAPK cascade in ß-cells, and Fig. 4
shows a speculative model of how extracellular signals may be
transduced through MAPK in ß-cells. Both glucose and PMA are reported
to increase the activity of the 44-kDa MAPK isoform in the INS-1
ß-cell line, but MAPK activation did not stimulate insulin secretion
(118). Similarly, activation of MAPK by inhibiting phosphotyrosine
phosphatases in rat islets of Langerhans did not stimulate insulin
secretion (117), while inhibition of MAPK activity by using PD098059 to
prevent MEK activation did not affect islet (287) or INS-1 cell (288)
secretory responses to nutrients, confirming that MAPK activation is
neither sufficient nor essential for regulated insulin secretion. If
the MAPKs are not involved in insulin secretion, they may be involved
in other ß-cell responses. Induction of early response genes
(junB, nur77, and zif268) in INS-1
cells is associated with MAPK activation (118), perhaps suggestive of a
role for MAPKs in proliferative responses. If ß-cell MAPKs are
involved in the regulation of proliferation, then the reported
differences in the activation of the 44-kDa MAPK between INS-1 cells
(118) and rat islets of Langerhans (117) may reflect the different
proliferative states of ß-cell lines and ß-cells in adult islets.
The activation state of the 44-kDa MAPK may therefore provide a future
target for the experimental manipulation of ß-cell proliferation,
which could have important therapeutic implications.
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E. Protein tyrosine kinases
There is relatively little information available at present on the
regulation of PTK activities in pancreatic ß-cells by insulin
secretagogues, although some PTK substrates have been identified, and
the speculative model shown in Fig. 4 contains both receptor and
cytosolic PTKs. The insulin receptor is probably one of the most
studied mammalian PTKs, and the recent detection of insulin receptor
mRNA and of the insulin receptor substrate (IRS-1) protein in ß-cells
(290) implies that insulin may modulate ß-cell function by autocrine
interactions, a notion that has not been considered seriously since the
report over a decade ago that normal ß-cells do not express
high-affinity receptors for insulin (291). In ßTC3 cells and rat
islets, glucose or insulin-like growth factor I enhanced the tyrosine
phosphorylation of a 97-kDa protein identified as the insulin receptor
ß-subunit (127) and of the 170/180 kDa insulin receptor substrates
IRS-1 and IRS-2 (124, 127), while glucose enhanced tyrosine
phosphorylation of an unidentified 125-kDa substrate (128).
The consequences to the secretory process of increased changes in the
tyrosine phosphorylation state of islet proteins are not always clear.
Receptor-mediated activation of PTKs either stimulates (292) or has no
effect on (126, 293) insulin release from cell lines and neonatal or
adult islets. An alternative approach is to enhance tyrosine
phosphorylation of a wide range of islet proteins by inhibiting
tyrosine phosphatases with vanadate or tungstate (117, 294). In a
recent study using mouse islets, this was accompanied by increased
production of inositol phosphates and inositol phospholipids, effects
that were attributed to increased tyrosine phosphorylation and
activation of PLC and phosphatidylinositol 3-kinase, respectively
(125). However, the vanadate-induced inhibition of tyrosine
phosphatases can be dissociated from insulin secretion. Under
conditions in which vanadate caused marked increases in the tyrosine
phosphorylation state of a number of islet proteins, it had no effect
on basal insulin secretion (117, 294), although higher concentrations
of vanadate did cause some enhancement of glucose-induced insulin
secretion, an effect that was attributed to alterations in membrane
potential, PI metabolism, and Ca2+-handling in the
ß-cells (294).
To date, most studies of the functional significance of PTK activation
and tyrosine phosphorylation have been based on inhibitors of PTK
activities and these have, as always, produced conflicting results.
Such studies have produced some evidence that PTKs are involved in
receptor-operated modulation of insulin secretion: the PTK inhibitor
genistein has been reported to prevent the inhibitory effects of
interleukin-1ß on insulin release (295), probably by inhibiting the
activation of nuclear factor 
B (296) and reducing NO production
(295, 297, 298).
The use of inhibitors has not produced a consensual view of the involvement of PTKs in ß-cell responses to nutrients. Thus, the PTK inhibitor genistein had stimulatory effects on insulin secretion from INS-1 cells (299), HIT-T15 cells (300), neonatal rat islets (301), and adult mouse islets (302), suggesting that PTKs exert a tonic inhibitory effect on the secretory process. However, PTK inhibitors have also been reported to have no effect on secretion (300), to inhibit glucose-stimulated insulin secretion from HIT-T15 cells (300) and rat islets (303), to inhibit CCh and glucose-induced insulin secretion from ßTC3 cells (128), and to inhibit GLP-1 and glucose-stimulated secretion from RIN 1046–38 cells (186). Some of the confusion undoubtedly arises from nonspecific effects of the inhibitors being used. PTK inhibitors have diverse effects on metabolic processes unrelated to the inhibition of PTK activity (304), and it has been suggested that the stimulation of insulin release by genistein is unrelated to inhibition of PTKs (302). As for the protein serine/threonine kinases, interpretations of the physiological functions of PTKs based solely on the use of inhibitors should be viewed with caution.
Investigations in this area are currently limited by a lack of information on the precise nature of the PTKs expressed in ß-cells and of the signal transduction mechanisms by which they are regulated. The activation of PTKs in response to physiological insulin secretagogues and the potential roles of PTKs in regulating ß-cell secretory and proliferative responses merit much more detailed study, since these pathways may offer potential for therapeutic intervention.
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V. Protein Phosphatases and Protein Dephosphorylation in ß-Cells |
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A. Serine/threonine protein phosphatases
Serine/threonine-PPs have broad and overlapping substrate
specificities, so their classification has generally been based both on
substrates and on inhibitors of the enzymes. Using such criteria, four
major serine/threonine-PPs have been identified (310). Type 1 PPs (PP1)
specifically dephosphorylate the ß-subunit of phosphorylase kinase
and are inhibited by nanomolar concentrations of the thermostable
proteins, inhibitor-1 (I-1) and inhibitor-2 (I-2) (310). Type 2 PPs
(PP2), which preferentially dephosphorylate the -subunit of
phosphorylase kinase, are unaffected by the PP1 inhibitor proteins and
are further subdivided into three groups of enzymes. PP2A, like PP1,
does not have an absolute requirement for divalent cations for its
activation, although PP2B and PP2C are dependent on
Ca2+/CaM and Mg2+, respectively. Two
heat-stable inhibitor proteins of PP2A have been purified from bovine
kidney: they show no homology to I-1 and I-2, are products of distinct
genes, and show negligible inhibitory activity toward PP1, PP2B, and
PP2C (314). PP1 and PP2A are homologous and highly conserved proteins
with catalytic subunits of molecular mass 36–37 kDa. The catalytic
subunit of PP2B shows 40% homology to those of PP1 and PP2A and is
much larger (61 kDa). PP2B is a heterodimer, consisting of the
catalytic domain coupled to a CaM-binding subunit. PP2C is unrelated in
structure to the other enzymes, and this is taken as evidence of two
distinct gene families. At least two isoforms of each
serine/threonine-PP are known to be present in mammalian cells (see
Ref. 310).
B. Protein phosphatases in ß-cells
Islets and ß-cell lines express several types of protein
phosphatase (Table 1). Early reports of Ca2+-stimulated PP
activity in homogenates or subcellular fractions prepared from rat
islets (315, 316, 317) were presumably measuring the activity of PP2B and
have been supported by the immunological detection in rat islets and
MIN6 ß-cells of calcineurin/PP2B (318, 319, 320) and of enzymically active
PP2B in MIN6 ß-cells (320). Enzyme activities and immunoreactivities
corresponding to PP1 and PP2A have also been detected in mouse and rat
islets and in RINm5F cells (319, 321, 322). There are no reports yet of
the detection of PP2C in islets or ß-cells.
It is not yet known whether PP1 and 2A are constitutively active in ß-cells, or whether their activities can be regulated. In other tissues there is some evidence that the activity of PP1 is indirectly inhibited by cAMP through the activation of PKA, which phosphorylates I-1, greatly increasing its inhibitory potency for PP1 (305). In contrast, early studies using islet homogenates detected a cAMP-stimulated PP activity (67, 315), although cAMP and cyclic GMP were later reported to have no effect on PP activity in islet homogenates (316, 317). More recently, sulfonylureas have been reported to cause a dose-dependent inhibition of PP1/2A activities in islet homogenates (323). There have been a number of experiments examining the effects of insulin secretagogues on PP activation in intact ß-cells and islets. In studies using intact RINm5F cells, forskolin did not consistently affect PP1/2A activities (324), although other studies using intact tissues have suggested that insulin secretagogues may affect the activities of PP1 and PP2A (320, 324, 325). Glucose is reported to cause a transient increase in PP1/2A activities in islets (320, 325), although arginine and KCl have been reported to decrease PP1/2A activities in RINm5F ß-cells (324). It has also been reported that carboxymethylation of the catalytic subunit of PP2A, associated with increased catalytic activity, resulted in an inhibition of nutrient-stimulated insulin secretion, suggestive of a negative modulatory role for PP2A in the insulin-secretory process (326).
Most of the evidence implicating PPs in ß-cell secretory responses has been obtained using PP inhibitors, and the same caveats should be applied to these studies as to studies using inhibitors of protein kinases (Section IV). Okadaic acid (OA) is a C38 polyether fatty acid inhibitor of PP1 and PP2A activities, which has been used to demonstrate the existence of substrate proteins for PP1 and 2A in rat islets (327). It has been proposed that one of these is the voltage-activated L-type Ca2+ channel, since in RINm5F cells OA induced increased Ca2+ influx through these channels (328), and this was accompanied by increased insulin secretion (328, 329). However, OA is also reported to stimulate insulin secretion from permeabilized islets maintained at a substimulatory Ca2+ concentration (327) and to enhance exocytosis in single ß-cells with minimal effects on Ca2+ current (321), suggesting that it has effects on the secretory system other than enhancing Ca2+ influx. The picture is further complicated by observations in rat islets that OA does not affect basal insulin release, but inhibits glucose-stimulated insulin release (319, 327), suggesting that PP1 and/or PP2A activities are required for stimulated insulin secretion.
Given the many potential sites of action of protein kinases in the secretory pathway (Section III.B), and the broad substrate specificities of PP1 and PP2A, it seems likely that global inhibition of PP1/2A activities would affect many stages of the stimulus-secretion pathway, and the disparate effects of OA on the secretory process could be encompassed by a model in which PP1 and/or PP2A have several sites of action, including 1) An early excitatory/permissive effect in secretory responses of whole cells to nutrients, the inhibition of which by OA would result in the inhibition of nutrient-induced secretion. 2) An inhibitory effect close to the point of depolarization-induced Ca2+ entry, the blocking of which would enhance Ca2+ influx and insulin release. This could be a direct effect on a VDCC subunit. 3) An inhibitory effect at a more distal site(s) in the stimulus-secretion coupling pathway, the blocking of which would result in enhanced basal and stimulated secretory responses. This could be via one or more of the proteins involved in secretory granule docking and fusion. The apparent discrepancy in responses of RINm5F cells between studies (328, 329) may reflect differences in the relative importance of these putative sites in a glucose-insensitive cell line.
Inhibitors have also been used to investigate the role of the Ca2+-dependent PP2B in secretory responses to Ca2+-mobilizing stimuli. Cyclosporin A (CsA) inhibited PP2B activity and increased protein phosphorylation in MIN6 cells, and this was accompanied by enhanced secretory responses of MIN6 and HIT-T15 ß-cells to glucose, tolbutamide, and KCl (320). In contrast, CsA was reported to have no effect on either basal or glucose-stimulated insulin secretion from rat islets, although the efficacy of CsA was not confirmed by measurements of PP2B activity in this study (319). The PP2B inhibitors, deltamethrin and calcineurin autoinhibitory peptide, are both reported to abolish the inhibitory effects of somatostatin and galanin on Ca2+-induced capacitance increases in single mouse ß-cells (330), suggesting that PP2B activation and the consequent dephosphorylation events are important in the inhibition of insulin secretion by receptor-mediated agonists. If substantiated, these observations imply a pivotal role for the phosphorylation event regulated by somatostatin and galanin being at a late stage of the secretory process (Section III.B). PP2B has also been implicated in the regulation of gene transcription in ß-cells. cAMP response element-mediated gene transcription in HIT cells was inhibited by CsA and another PP2B inhibitor, FK506 (331, 332), and AVP has been reported to induce CsA- and FK506-sensitive phosphorylation and activation of the CRE-binding protein in these cells (333).
Since protein phosphorylation is an important process in the regulation
of insulin secretion, protein dephosphorylation via PPs is required,
either to switch off linear transduction cascades or to maintain the
activity of cycling systems (Figure 5).
The important, and as yet largely unanswered, question is whether the
PPs themselves offer a point of regulation. Future studies directed
toward whether, and through what mechanisms, physiologically relevant
insulin secretagogues modulate the activities of ß-cell PPs should go
a long way toward answering this question.
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VI. Protein Kinase-Independent Secretory Pathways |
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Studies using selective kinase inhibitors indicate that PKA and PKC are the sole response elements in the stimulation of insulin secretion by cAMP and DAG analogs, respectively (137, 163, 198). These observations suggest that kinase activation is indeed obligatory for secretory responses to those receptor-operated nonnutrients that enhance insulin secretion primarily through activating AC or PLC. In contrast, some studies using CaMK inhibitors or CaM antagonists suggest that increases in cytosolic Ca2+ may stimulate insulin secretion through mechanisms other than the activation of CaMKs. Thus, Ca2+-induced insulin secretion from permeabilized ß-cells was not inhibited by high concentrations of the CaM antagonists W7 and TFP (334) or by the CaMK II inhibitor KN-62 (228); the CaM inhibitor calmidazolium attenuated stimulus-dependent increases in ß-cell Ca2+ but stimulated insulin secretion (335); and KN-62 and the inhibitory peptide CaMK290–309 produced only partial (55–60%) inhibition of Ca2+-induced capacitance increases in mouse ß-cells (164). These observations implicate the involvement of a Ca2+-sensitive protein(s), in addition to CaMK II, in generating secretory responses to Ca2+. Pancreatic ß-cells express many Ca2+-sensitive proteins that may be involved in signal transduction, although a detailed analysis of their functions is beyond the scope of this review. However, on current evidence, likely candidates include the 100-kDa cPLA2 (123, 336, 337, 338), which generates AA upon Ca2+-dependent activation; and the SNARES, such as VAMP and SNAP-25, which are thought to be involved in the Ca2+-dependent docking and/or fusion of insulin-containing secretory granules at the site of exocytosis (182, 184, 186).
The existence of a kinase-independent component of the secretory response to Ca2+ could explain the commonly reported, but seldom commented upon, observation that protein kinase inhibitors often do not fully inhibit secretory responses to the nutrient secretagogues that operate primarily by increasing intracellular Ca2+. For example, the kinase inhibitors TFP (20 µM) (225), chlorpromazine (100 µM) (81), dibucaine (500 µM) (81), H-7 (50 µM) (268), clomiphene (50 µM) (272), and KN-62 (10 µM) (229, 230) all produced a less than total inhibition of glucose-induced insulin release. Furthermore, several studies using staurosporine, a relatively nonselective kinase inhibitor, have demonstrated that the inhibition of glucose-induced insulin secretion was not complete even in the presence of supramaximal concentrations of staurosporine (89, 270, 271, 283). The simplest explanation for these observations is that kinase activation is an important part of normal secretory responses to nutrients, but that it cannot alone account for the full secretory response to Ca2+-mobilizing nutrient stimuli. It is unclear at present whether the kinase-dependent, inhibitor-sensitive component of the response reflects insulin release from a different pool of secretory granules to the kinase-independent component, or whether it is an earlier stage in a single secretory process, such as moving the secretory granules to a release site, or rendering the granules competent for exocytosis. Identifying the components of the kinase-independent regulatory pathways will lead to a better understanding of the secretory process and may identify new therapeutic targets through which to manipulate insulin release.
Under certain experimental circumstances it is possible to produce an apparently regulated insulin-secretory response without any requirement for kinase activation, by exposing ß-cells to the amphiphilic peptide mastoparan (MP) (339, 340, 341, 342, 343), to the neuronal protein myelin basic protein (MBP) (344, 345), or to the unsaturated fatty acid AA (261, 346). Although they are structurally very different, the effects of these agents on the ß-cell are similar in many respects. Thus, they initiate secretory responses that do not require an influx of Ca2+, are not ATP-dependent, and are resistant to a wide variety of protein kinase inhibitors (340, 341, 342, 344). Their similar effects imply a similar mode of action, and this may be by generating, or acting as, fusogens at the last stage in exocytosis. MBP associates with islet cell membranes (345), where it may act as a direct fusogen or generate other fusogens. MP has been shown to interact with membrane-associated low molecular mass and heterotrimeric G proteins (347, 348). In addition, MP stimulated the GTPase activity in insulin-secretory granules (343), and both this effect and that on the stimulation of insulin secretion were blocked by pertussis toxin (339, 341, 343). Finally, MP-induced insulin secretion was blocked by inhibitors of phospholipases, the class of enzymes that generate AA (339, 341). These observations are consistent with a model in which MP (and perhaps MBP) stimulates insulin secretion by activating a G protein-coupled phospholipase, perhaps on the secretory granule membrane, to generate a fusogenic substance such as AA. The effects of substances such as MP, MBP, and AA are unlikely to be totally specific and in any case are sufficiently nonphysiological to merit some degree of caution in extrapolating their mode(s) of action to those of physiological secretagogues. However, there is also evidence that glucose can regulate insulin secretion through a mechanism that is independent of changes in membrane potential or changes in cytosolic Ca2+, and which does not require the activation of PKA or PKC (349). The nature of this regulatory mechanism and its physiological relevance are as yet uncertain, but a fuller understanding should provide insights into the secretory process in general and into the importance of regulated kinases in that process.
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VII. Summary and Future Perspectives |
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) in
which Ca2+-regulated protein kinases are involved in the
initiation of insulin-secretory responses by the physiologically
important nutrient secretagogues, although other
Ca2+-sensitive response elements may also be required for a
full secretory response. In this consensus model, kinases from the PKA
and PKC families are vitally important for the modulation of secretory
responses by receptor-operated, nonnutrient secretagogues, but their
involvement in nutrient-induced insulin secretion is still a matter of
debate. Definitive evidence is not yet available about the involvement
of other kinase transduction pathways in the regulation of insulin
secretion, including the MAPK cascade and those operating through
tyrosine kinases/phosphatases.
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-isoform (63) may have access to areas denied the
full-length enzymes. In this case the existence of similar isoforms
from independent genes may simply reflect a fail-safe adaptation to
ensure the expression of active and regulatable CaMK activity in the
event of deleterious mutations of the gene encoding one isoform. The
isoforms of PKA are also subject to the same mechanism of regulation of
enzymic activity, in this case by the availability of cAMP, but the
subcellular localization of PKA activity is determined by the isoform
of the regulatory subunit within the holoenzyme (Section
II.B). The expression of PKA isoforms, or at least those of the
regulatory subunit, may therefore exist to subserve specific functions,
and the regulation of activity, substrate specificity, and cellular
function may be determined as much by cellular location as by the
isoform structure. The PKC family of isoforms differs from CaMK and PKA
in that subgroups of isoforms are subject to different mechanisms of
regulation and may be confined to different intracellular compartments
(Section II.C). One explanation for this diversity is that
the subgroups of PKC isoforms (conventional, novel, atypical) have
evolved to subserve specific and different functions within ß-cells,
in which case experimental studies of PKC function should be directed
toward individual isoforms, or at least isoform subgroups, to produce
interpretable information. Substantial insights into protein kinase
function in ß-cells will be dependent upon the development of novel
methods for the detection, activation, and inhibition of individual
protein kinase isoforms.
Despite the overwhelming evidence of kinase involvement in the
secretory process, we are still largely ignorant of the identity and
functions of the kinase substrates (Section III.B), and this
ignorance highlights another important question for future studies:
is there a pivotal phosphoprotein, or a group of phosphoproteins,
through which all kinase-activating external stimuli converge to
influence the secretory process? The lack of a definitive answer
to this question at present is probably due to limitations in current
experimental approaches. Attempts to detect important substrates by
measuring changes in phosphorylation of endogenous proteins may detect
transduction elements in which the mass of phosphoprotein increases
(Figure 5, upper panel) but will not necessarily detect
changes in the flux of phosphate through the substrate, which may be
equally important (Figure 5, lower panel). More immediately
useful information can be gained by adopting the "candidate
protein" approach in which the phosphorylation of substrates known to
be involved in transduction cascades is measured, although this
approach is inherently unlikely to identify novel pathways or
mechanisms. The rapid recent advances in cell and molecular biology are
presenting ever more candidate proteins for this approach, and our
ultimate understanding of the role of protein phosphorylation in the
regulation of insulin secretion is probably dependent upon the
application to ß-cells of conceptual advances made in more
experimentally accessible tissues.
As the sequencing of the human genome proceeds ever more rapidly, current estimates suggest that it may contain coding sequences for more than 1000 protein kinases. The 361 studies referred to in this review have produced only limited insight into the function of a few of these enzymes in pancreatic ß-cells. There is clearly much work to be done.
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Footnotes |
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References |
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TopAbstractI. IntroductionII. Protein Kinases in...III. Phosphorylation of...IV. Protein Kinases and...V. Protein Phosphatases and...VI. Protein Kinase-Independent...VII. Summary and Future...References |
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