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Protein Kinases, Protein Phosphorylation, and the Regulation of Insulin Secretion from Pancreatic ß-Cells.

Cellular and Molecular Endocrinology Group, Biomedical Sciences Division, King’s College London, Campden Hill Road, Kensington, London, W8 7AH, United Kingdom

	Abstract

Top Abstract I. Introduction II. Protein Kinases in... III. Phosphorylation of... IV. Protein Kinases and... V. Protein Phosphatases and... VI. Protein Kinase-Independent... VII. Summary and Future... References

 

I. Introduction

II. Protein Kinases in ß-Cells: Expression and Characteristics

A. Ca²⁺/calmodulin-dependent protein kinases

B. Cyclic nucleotide-dependent protein kinases

C. Ca²⁺/phospholipid-dependent protein kinases

D. Mitogen-activated protein kinases

E. Protein tyrosine kinases

III. Phosphorylation of Endogenous Proteins in ß-Cells

A. Methods for studying protein phosphorylation in ß-cells

B. Endogenous kinase substrates in ß-cells

IV. Protein Kinases and the Regulation of Insulin Secretion

A. Ca²⁺/calmodulin-dependent protein kinases

B. Cyclic nucleotide-dependent protein kinases

C. Ca²⁺/phospholipid-dependent protein kinases

D. Mitogen-activated protein kinases

E. Protein tyrosine kinases

V. Protein Phosphatases and Protein Dephosphorylation in ß-Cells

A. Serine/threonine protein phosphatases

B. Protein phosphatases in ß-cells

VI. Protein Kinase-Independent Secretory Pathways

VII. Summary and Future Perspectives

	I. Introduction

Top Abstract I. Introduction II. Protein Kinases in... III. Phosphorylation of... IV. Protein Kinases and... V. Protein Phosphatases and... VI. Protein Kinase-Independent... VII. Summary and Future... References

 
IT IS now well established that the secretion of insulin by pancreatic ß-cells is regulated primarily by the circulating concentrations of nutrient secretagogues, including glucose, fatty acids, and some amino acids, and that these agents are unusual among endocrine stimuli because they influence the secretory process as a result of their metabolism within ß-cells rather than by actions at cell surface receptors. Nutrient stimuli are responsible for initiating insulin secretion from ß-cells, but nutrient-induced secretory responses can be radically modified by nonnutrient secretagogues. These include a wide variety of hormones and neurotransmitters that act at specific cell surface receptors that are linked, via GTP-binding proteins, to intracellular effector systems. Coupling from receptors to effectors through G proteins in ß-cells appears to be similar in all important respects to that reported in other tissues, and there have been many excellent reviews of this area recently (1, 2, 3, 4).

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⁺ (K_ATP) 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 K_ATP 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.

	II. Protein Kinases in ß-Cells: Expression and Characteristics

Top Abstract I. Introduction II. Protein Kinases in... III. Phosphorylation of... IV. Protein Kinases and... V. Protein Phosphatases and... VI. Protein Kinase-Independent... VII. Summary and Future... References

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 (K_a) = 10 µM], CaM (K_a = 2 nM), myosin light chains [Michaelis-Menton constant (K_m) = 70 µM], and ATP (K_m = 70 µM), and a susceptibility to inhibition by trifluoperazine [TFP, inhibition constant (K_i) = 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 K_a for cAMP of 0.01 µM, a K_m 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 K_a for cAMP of 0.08 µM, and a K_m 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 K_m 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 (K_a 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, PKI_6–22, 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 K_m for ATP of 10 µM, K_a for Ca²⁺ of 3.9–10 µM, K_a for PS of 12–18 µg/ml, K_a for diolein of 2.5 µg/ml, and K_m 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).

	III. Phosphorylation of Endogenous Proteins in ß-Cells

Top Abstract I. Introduction II. Protein Kinases in... III. Phosphorylation of... IV. Protein Kinases and... V. Protein Phosphatases and... VI. Protein Kinase-Independent... VII. Summary and Future... References

 
Identification of kinase substrates and of their cellular functions is crucial to a full understanding of the regulatory roles of protein kinases, but this is perhaps the area of most uncertainty at present. Numerous kinase substrates have been reported in ß-cells, but little functional information is available and, until recently, the vast majority of these proteins were identified only by their apparent molecular mass or isoelectric point determined by their migration positions on one-dimensional or two-dimensional PAGE. This paucity of information has several causes, some specific to ß-cells and some with more general implications. Thus, it is difficult to obtain sufficient quantities of purified islets of Langerhans from experimental animals to permit the isolation, identification, purification, and study of particular kinase substrates of interest. Insulin-secreting cell lines are available in larger amounts, but they are not ideal models for studying ß-cell kinase function because cell lines may express different proteins and may have different secretory responses to those of authentic ß-cells. More generally, protein phosphorylation is a common regulatory mechanism in eukaryotic cells, and a lack of knowledge about the precise mechanisms of secretory granule trafficking and exocytosis in ß-cells makes it difficult to determine which of the many reported phosphorylation events may be involved in the regulation of secretion and which are involved in other cellular processes.

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 K_ATP 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 uK_ATP-1, K_ATP-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 K_ATP 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 K_ATP 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 G_i 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 G_i 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).

	IV. Protein Kinases and the Regulation of Insulin Secretion

Top Abstract I. Introduction II. Protein Kinases in... III. Phosphorylation of... IV. Protein Kinases and... V. Protein Phosphatases and... VI. Protein Kinase-Independent... VII. Summary and Future... References

 
It should be stated clearly at the beginning of this section that much of the evidence implicating particular protein kinases in physiologically relevant insulin-secretory responses is inconclusive, confusing, and often contradictory. In our view, much of the confusion is caused by studies of kinase function that are based on measuring secretory responses to a single protein kinase inhibitor of uncertain specificity. A full discussion of protein kinase inhibitors is beyond the scope of this review but, given the importance of this experimental approach in shaping the consensus view of ß-cell function, it is worth considering briefly the different types of protein kinase inhibition that have been used in pancreatic ß-cells.

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 G_s (210), or by overexpressing a constitutively active -subunit of G_s (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.

View larger version (23K): [in this window] [in a new window]   Figure 1. The role of CaMK in ß-cell responses to nutrient and nonnutrient stimuli. The schematic diagram shows how CaMK II may be involved in transducing signals from nutrients (e.g., glucose) and nonnutrients (e.g., acetylcholine, ACh). Nutrients: glucose enters the ß-cell on the GLUT2 transporter and is metabolized with a consequent generation of ATP and closure of KATP channels. The decreased efflux of K+ leads to depolarization of the ß-cell with the consequent opening of VDCCs and an influx of extracellular Ca2+ down its concentration gradient. The Ca2+ binds to CaM, and the Ca2+/CaM complex binds to the inactive kinase (CaMKi), which becomes enzymically active (CaMKa). The active CaMK phosphorylates (PO4) ß-cell substrate proteins, and these phosphorylation events stimulate the secretory process. Nonnutrients: agonists bind to cell-surface receptors that are coupled via the heterotrimeric GTP-binding protein Gq to PLC. Receptor occupancy activates PLC with the consequent generation of IP3 by the hydrolysis of membrane inositol phospholipids. IP3 releases stored Ca2+ from the endoplasmic reticulum, and the elevation in intracellular Ca2+ activates CaMK, as described above.

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 G_s 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.

Unt