COMMUNICATION:
Translocation Inhibitors Define Specificity of Protein Kinase C Isoenzymes in Pancreatic beta -Cells*

(Received for publication, May 29, 1996, and in revised form, November 1, 1996)

Michael Yedovitzky Dagger , Daria Mochly-Rosen §, John A. Johnson §, Mary O. Gray §, Dorit Ron §, Eva Abramovitch Dagger , Erol Cerasi Dagger and Rafael Nesher Dagger

From the Dagger  Department of Endocrinology and Metabolism, Hebrew University-Hadassah Medical Center, 91120 Jerusalem, Israel and the § Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94035

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific <UNL>r</UNL>eceptor for <UNL>a</UNL>ctivated <UNL>C</UNL> <UNL>k</UNL>inase (RACK) (Mochly-Rosen, D. (1995) Science 268, 247-251) that provides an anchoring site in close proximity to the isoenzyme's specific substrate. Pancreatic islet cells contain at least six PKC isoenzymes (Knutson, K. L., and Hoenig, M. (1994) Endocrinology 135, 881-886). Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, alpha PKC and epsilon PKC translocate to the cell's periphery, while delta PKC and zeta PKC translocate to perinuclear sites. beta C2-4, a peptide derived from the RACK1-binding site in the C2 domain of beta PKC, inhibits translocation of alpha PKC and reduces insulin response to glucose. Likewise, epsilon V1-2, an epsilon PKC-derived peptide containing the site for its specific RACK, inhibits translocation of epsilon PKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both alpha PKC and epsilon PKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of alpha PKC but not epsilon PKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.


INTRODUCTION

Protein kinase C is a family of 11 lipid-dependent serine/threonine kinases involved in a wide spectrum of signal transduction (7, 8). Upon activation, PKC1 isoenzymes translocate to new cellular sites, including the plasma membrane (9, 10), cytoskeletal elements (11, 12), and the nucleus (13, 14), as well as other subcellular compartments (15). Many cells are known to contain several isoenzymes (16, 17), each localizing to a different cellular site upon stimulation (18). The multiplicity of isoforms of a single enzyme renders the analysis of enzyme-function relationship difficult. Recent work revealed that activated PKC isoenzymes bind anchoring proteins termed RACKs (1-3), believed to be positioned in close proximity to the isoenzyme's substrate. It was further shown that the functional specificity of the PKC isoenzyme is determined, in part, by the differential localization of the isoenzyme-specific RACKs (19). The RACK for beta PKC, RACK1, has been cloned, and at least part of its binding site on beta PKC has been mapped to a short sequence within the C2 domain (1). beta C2-4, a nonopeptide derived from this region, inhibits phorbol ester-induced translocation of the C2-containing isoenzymes but not the translocation of C2-less isoenzymes such as delta - and epsilon PKC when tested in intact cells (1). A short peptide derived from the V1 region of epsilon PKC, epsilon V1-2, was similarly shown to inhibit the translocation of epsilon PKC, but not alpha -, beta -, and delta PKC (20). Furthermore, these isozyme-specific inhibitors blocked the specific function of individual isoenzymes; for example, epsilon V1-2, but not beta C2-4, inhibited phorbol 12-myristate 13-acetate-induced regulation of the contraction rate in intact cardiomyocytes. Here we use these novel PKC isozyme-specific inhibitors to determine that PKC activation is part of the signals involved in the regulation of glucose-induced insulin secretion and to identify the specific isoenzymes that mediate this glucose effect.


MATERIALS AND METHODS

Islets obtained from 200-g male Sprague-Dawley rats were cultured for 3-5 days in glass chamber slides coated with extracellular matrix of bovine corneal endothelial origin (21). When more than 75% of the islet cells spread out to form a monolayer, the media were replaced with modified Krebs Ringer solution (KRB) (22) containing either 2.5 or 20 mM glucose. Following a brief wash, the islets were fixed in cold acetone, blocked with 1% normal goat serum for 1 h, and treated overnight with the specific anti-PKC isoenzyme antibody (Research and Diagnostic Antibodies, Berkeley, CA). Fluorescein isothiocyanate-linked goat-anti-rabbit IgG (Sigma Israel Chemicals, Rehovot, Israel) was applied for 2 h, and the slides were mounted in 90% glycerol, 10% phosphate-buffered saline, 0.1% sodium azide, 3% diazabicyclo[2.2.2]octane, pH 9.0, for microscopic imaging. Histochemical imaging was conducted on a PhioBos 1000 confocal microscope (Sarastro Inc., Ypsilanti, MI), equipped with Zeiss Universal Optics and argon laser illumination. The anti-PKC antisera each exhibited a single band of the appropriate size on Western blot (10 µg of rat brain or rat islet homogenate/lane). No bands were observed when the antibodies were preincubated with excessive amounts of their corresponding antigen derived from the V5 region of the isoenzyme (PKC-epsilon-(728-737) or PKC-alpha -(Tyr663-(664-672)), Research and Diagnostic Antibodies, Berkeley, CA). Preincubation of anti-alpha PKC with excess PKC-alpha -(Tyr663-(664-672)) and anti epsilon PKC with excess PKC-epsilon-(728-737) completely abolished the fluorescence images of the glucose-dependent isoenzyme translocation. The cells were identified as beta -cells by counterstaining with tetramethylrhodamine B isothiocyanate-conjugated rabbit anti-guinea pig IgG and guinea pig anti-insulin serum (Dako Corp., Carpinteria, CA).

Freshly isolated islets were used for insulin release studies. Islets were preincubated for 1 h in KRB-BSA buffer containing 2.5 mM glucose, then transferred, 1-2 islets per tube, for an additional 60-min incubation in KRB-BSA buffer containing either 2.5 mM or 20 mM glucose as described previously (22). When calcium-free buffer was used, 5 mM EGTA was added to the calcium-free KRB-BSA buffer during the test period.

Peptides were synthesized at the Hebrew University School of Medicine Intradepartmental Equipment Services. Peptide introduction into islet cells was achieved by transient permeabilization ("skinning") (4). Ten-minute skinning, followed by a 1-h incubation of isolated rat islets with Krebs buffer containing 2.5 mM glucose, had no effect on islet viability as examined by trypan blue exclusion or by the exhibition of intact islet secretory response to glucose in either batch or perifusion studies.

Insulin release was measured by radioimmunoassay using specific guinea pig anti-rat insulin antiserum (Linco Research, St Charles, MO) and rat insulin standard (Novo Research Institute, Bagsvaerd, Denmark) (22). Data presented are mean net insulin values after subtraction of non-stimulated level (2.5 mM glucose). Statistical significance was determined by paired, non-parametric comparison to control or to control skinned islet, using the Wilcoxon test.


RESULTS AND DISCUSSION

The role of PKC as an amplifier of the glucose-generated signal to release insulin has been well established (5, 23-25). Six PKC isoenzymes, alpha , beta , delta , epsilon , zeta , and iota , were found thus far in rat pancreatic islets of Langerhans (5, 6, 25). The specific function of the individual isoenzymes remains unknown. We used adult rat islet cultures (21) to assess the direct effect of glucose on PKC isoenzymes in intact islet cells. Site-specific translocation could be demonstrated for alpha -, delta -, epsilon -, and zeta PKC (Fig. 1). Confocal microscopy imaging revealed that alpha PKC and epsilon PKC redistributed following glucose stimulation to the cell's periphery, delta PKC concentrated in an asymmetric structure in perinuclear region (possibly the Golgi apparatus), and zeta PKC concentrated as a ring around the cell nucleus (Fig. 1).


Fig. 1. Glucose-dependent translocation of protein kinase C isoenzymes in rat islets of Langerhans. Islet monolayers were incubated for 60 min in either 2.5 mM (Basal) or 20 mM glucose (Stimulated), fixed in acetone, and stained with isoenzyme-specific antibodies for confocal imaging. alpha - and epsilon PKC translocated to the cell periphery while delta PKC concentrated asymmetrically in a perinuclear region and zeta PKC formed a ring structure around the cell nuclei. Color gradients depicted as black, blue, yellow, and red indicate, in increasing order, the relative concentration of the isoenzyme.
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Isoenzyme alpha  is a member of the calcium-sensitive cPKC subfamily (7, 8). Alterations in cytosolic calcium levels play a prominent role in beta -cell stimulus-secretion coupling for insulin release (for review, see, for example, Ref. 26). We therefore examined whether modulation of the calcium-sensitive PKC subtypes affects glucose-induced insulin release in rat islets. beta C2-4 (Ser-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr) is a nonopeptide derived from the binding site (amino acids 218-226) of beta IIPKC to RACK1 (19), a well conserved sequence within the C2 domain of members of the cPKC subfamily. Introduction of beta C2-4 into islet monolayers by means of transient permeabilization at low temperature (skinning) (4) abolished the glucose-induced translocation of alpha PKC to the cell's periphery (Fig. 2c), whereas beta C2-4-s, a scrambled control peptide (Trp-Asn-Pro-Glu-Ser-Leu-Asn-Thr), did not prevent translocation (Fig. 2d).


Fig. 2. Inhibition of glucose-dependent translocation of alpha PKC by beta C2-4, a cPKC-specific translocation inhibitor. Confocal images are shown of control skinned islet cultures stained with anti-alpha PKC following a 60-min incubation in KRB containing 2.5 mM glucose (a), skinned islet incubated in KRB containing 20 mM glucose (b), skinned islet treated with beta C2-4 then incubated in KRB containing 20 mM glucose (c), or skinned islet treated with the control scrambled peptide beta C2-4-s and incubated in KRB containing 20 mM glucose (d). e, inhibition of glucose-stimulated insulin release by beta C2-4 in isolated rat islets. Insulin response was determined following a 60-min stimulation with 20 mM glucose in untreated islets (Control), control skinned islets without peptide addition (Skinned), skinned islets in the presence of 10 µM beta C2-4 (beta C2-4), or skinned islets in the presence of 10 µM scrambled peptide (beta C2-4-s). * denotes statistical significance p < 0.02.
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Moreover, administration of beta C2-4 into freshly isolated islets resulted in 35% reduction in the insulin response to glucose stimulation (Fig. 2e); introduction of the scrambled analog, beta C2-4-s, had no inhibitory effect on insulin secretion. To rule out the possibility that the partial inhibition of the insulin response was the result of unequal penetration of the peptide, freshly isolated islets in suspension were skinned in the absence or presence of 10 µM beta C2-4 and subsequently incubated with 20 mM glucose. At the end of a 60-min stimulation, the islets were fixed in paraformaldehyde, dehydrated in ethanol, and further treated in propylene oxide Surre mixture. Following resin polymerization, 5- and 10-µm slices were prepared and stained with anti-alpha PKC antibodies. Confocal imaging revealed uniform inhibition of the glucose-induced isoenzyme translocation throughout the sections (not shown), indicating homogeneous penetration of the octapeptide throughout the islet. beta C2-4 has been shown to be equally effective against all members of the classical PKC subfamily (19). While alpha PKC often has been reported to be the predominant PKC isoenzyme in islet beta -cells, beta PKC exhibits only scant expression in these cells (5); we, however, assume that all cPKC isoenzymes were equally inhibited by beta C2-4. The fact that maximally effective concentrations of beta C2-4 had only a partial inhibitory effect on glucose-induced insulin release may therefore indicate that in addition to the cPKC subfamily, other PKC isoenzymes or non-PKC stimulus amplifiers are involved in the glucose-generated stimulus-secretion coupling.

Despite the pivotal role of cytosolic calcium mobilization in the control of insulin release, data are accumulating supporting the role of additional, calcium-independent signals in the modulation of insulin secretion (27-29). We therefore examined the role of epsilon PKC, a calcium-insensitive PKC isoenzyme in glucose-induced insulin secretion utilizing epsilon V1-2, the octapeptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) derived from the RACK-binding site of epsilon PKC (amino acids 14-21 within the V1 region of the isoenzyme) (20). Introduction of epsilon V1-2 into islet monolayers by means of skinning abolished glucose-induced translocation of epsilon PKC (Fig. 3c); the scrambled analog epsilon V1-2-s (Leu-Ser-Glu-Thr-Lys-Pro-Ala-Val) had no effect (Fig. 3d). Moreover, introduction of epsilon V1-2 into freshly isolated islets in suspension inhibited the insulin response to glucose by 40% (Fig. 3e), whereas epsilon V1-2-s, the scrambled analog, had no inhibitory effect (Fig. 3e). In histochemical studies, epsilon V1-2 had no effect on glucose-dependent translocation of alpha PKC and, vice versa, beta C2-4 had no effect on the translocation of epsilon PKC (not shown).


Fig. 3. Inhibition of glucose-dependent translocation of epsilon PKC by epsilon V1-2, a RACK2-binding peptide. Immunohistochemical images are shown of islet cultures skinned and stained with anti-epsilon PKC following a 60-min incubation in KRB containing 2.5 mM glucose (a), skinned islets incubated in KRB containing 20 mM glucose (b), skinned islets treated with epsilon V1-2 then incubated in KRB containing 20 mM glucose (c), or skinned islets treated with the control scrambled peptide epsilon V1-2-s then incubated in KRB containing 20 mM glucose (d). e, inhibition of glucose-induced insulin secretion by epsilon V1-2 in isolated rat islets. Insulin response was determined following a 60-min stimulation with 20 mM glucose in untreated islets (Control), control skinned islets without peptide added (Skinned), skinned islets in the presence of 10 µM epsilon V1-2 (epsilon V1-2), or skinned islets in the presence of 10 µM scrambled peptide (epsilon V1-2-s).* denotes statistical significance of p < 0.004 compared with glucose-stimulated control.
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5 µM was found to be a maximally effective concentration for both beta C2-4 and epsilon PKC in 1-h incubations, never exceeding 43% inhibition of glucose-induced insulin release. The effect of the two peptides was additive at that concentration in fresh islets incubated for 60 min at 20 mM glucose (Fig. 4b). The fact that both peptides together inhibited only 67% of the glucose-mediated insulin release suggests that alpha PKC and epsilon PKC are each independently involved in one of several distal coupling systems (see below). Stimulation of islet monolayer in the absence of Ca2+ (and in the presence of EGTA) abolished glucose-dependent translocation of alpha PKC but not of epsilon PKC (Fig. 4a). It also diminished the glucose-induced insulin response in fresh islets by 85% (Fig. 4b), providing further evidence that alpha PKC but not epsilon PKC is involved in the calcium-mediated regulatory signal to release insulin. However, addition of mannoheptulose, an inhibitor of glucose metabolism, completely abolished the glucose-induced translocation of both alpha - and epsilon PKC isoenzymes (Fig. 4a) as well as the islet insulin response to the sugar (Fig. 4b), indicating that the glucose-stimulatory coupling signal for the activation of PKC is linked to glycolysis, the primary signal for beta -cell insulin response (26).


Fig. 4. a, islet monolayer subjected to calcium-free (+5 mM EGTA) medium exhibits no glucose-dependent translocation of alpha PKC, as compared with glucose-stimulated control culture. Glucose-dependent translocation of epsilon PKC is not affected by calcium omission. In fresh islet, omission of calcium diminishes the insulin response to 1-h stimulation with glucose by 85% (b). Mannoheptulose (30 mM), a glycolytic inhibitor, impedes glucose-stimulated translocation of both alpha PKC and epsilon PKC (a). Addition of 30 mM mannoheptulose fully inhibits glucose-stimulated insulin response in fresh islets (b). In fresh islets, the inhibitory effect of beta C2-4 and epsilon V1-2 (5 µM each) on glucose-mediated insulin response is additive (b).* denotes statistical significance of p < 0.02 compared with glucose-stimulated control; ** denotes statistical significance p < 0.05 compared with either beta C2-4 or epsilon V1-2 alone; *** denotes statistical significance of p < 0.005 compared with glucose-stimulated control.
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The islet beta -cell coupling mechanism regulating the insulin secretory response to glucose is a complex sequence of metabolic events (for review, see, for example, Ref. 26), leading to a unique multiphasic dynamic of hormonal secretion (for review, see Ref. 30). While the primary coupling signal originates from glucose metabolism, leading to calcium mobilization and activation of poorly defined calcium-dependent pathways, the same primary signal also activates numerous distal potentiating pathways, some calcium-dependent, others calcium-independent (27-29). Since insulin secretion is strongly modulated by the duration of glucose stimulation (30), it is possible that different PKC isoenzymes have distinct roles in the different phases of release. This subject is presently under investigation. Islets coupling signals originating from activation of adenylate cyclase, phospholipase C, and phospholipase A2 are among the more thoroughly investigated potentiating signals involved in the modulation of the insulin response to glucose (31-33). Messengers generated from these key metabolic pathways are known to activate multiple PKA, PKC, and CaM kinases, each controlling a specific amplifying branch of the insulin stimulus-secretion coupling pathway, resulting in the multiphasic dynamics of hormonal secretion in response to glucose stimulus (30, 31). The identification of PKC isoenzyme-specific binding proteins offers novel tools to resolve the specific contribution of each isoform to this complex of interrelated signals. Furthermore, RACK-binding translocation inhibitors should prove to be valuable tools in resolving the specific function of the individual PKC isoenzyme in cells expressing multiple forms of the enzyme as well as in identifying their specific substrates.


FOOTNOTES

*   This work was supported in part by a grant from the JDF International (to R. N.) and from the Piccioto Foundation (to E. C.) and National Institutes of Health Grant HL43380 (to D. M.-R.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Dept. of Endocrinology and Metabolism, Hadassah University Hospital, P.O. Box 12000, 91120 Jerusalem, Israel. Tel.: 972-2-677-6787; Fax: 972-2-643-7940; E-mail: nesherr{at}cc.huji.ac.il.
1    The abbreviations used are: PKC, protein kinase C; KRB, Krebs Ringer bicarbonate; BSA, bovine serum albumin.

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