The influence of the insulin secretagogues, carbachol and glucose, on protein kinase C activation in isolated pancreatic islets has been examined.
THE JOURNAL OF BIOLOGICAL CHEMISTRY (~11990 by The American Society for Biochemistry
Vol. 265, No. 25, Issue of September 5, pp. 14938-14946, 1990 Printed in U.S. A.
and Molecular Biology, Inc.
Effects of Insulin Secretagogues on Protein Kinase C-catalyzed Phosphorylation of an Endogenous Substrate in Isolated Pancreatic Islets* (Received for publication, Richard Michael
A. Easom$#, McDaniel+
From the Departments Washington University
Michael
LandtSll,
Jerry
of SPathology and llPediatrics, School of Medicine, St. Louis,
R. Colcall,
**Division Missouri
The influence of the insulin secretagogues, carbachol and glucose, on protein kinase C activation in isolated pancreatic islets has been examined by determination of the phosphorylation state of an endogenous 80-kDa protein substrate of protein kinase C. The islet 80-kDa protein was identified as the myristoylated alaninerich C kinase substrate previously described (Stump0 D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012-4016) by immunoprecipitation studies. The muscarinic agonist, carbachol (500 PM), induced insulin secretion and a time-dependent increase in the phosphorylation state of this protein in islets. This phosphorylation was maximal (220 f 24% of control) at 5 min and was suppressed by the protein kinase C inhibitor, staurosporine. Concentrations of glucose (28 mM) which induce maximal insulin secretion did not induce a statistically significant increase in 80-kDa phosphorylation. The combination of carbachol and a submaximally stimulatory concentration of glucose (10 mM), when added simultaneously, exerted a marked synergistic effect on insulin secretion and a synergistic effect on the phosphorylation of the 80-kDa protein kinase C substrate. These data suggest that the activation of protein kinase C may play an important role in carbachol-induced insulin secretion and in the potentiation by carbachol of insulin secretion induced by glucose. However, the activation of protein kinase C does not appear to be a primary determinant of insulin secretion induced by glucose alone.
The possibility that protein kinase C activation may participate in glucose-induced insulin secretion from isolated pancreatic islets has generated considerable recent interest (for reviews see Refs. 1 and 2). Evidence in support of this possibility includes the observation that phorbol esters, potent activators of protein kinase C, promote insulin secretion both from isolated islets (3-7) and from insulin secreting clonal cell lines (8). Diacylglycerol, an endogenous activator of protein kinase C, also stimulates insulin secretion when added * This work was supported in part by a fellowship from the Juvenile Diabetes Foundation International (to R. A. E.) and by National Institutes of Health grants DK-06181 (to M. L. M.), DK-34388, and DK-01553 (to J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. 5 To whom correspondence should be addressed: Dept. of Pathology, Washington University School of Medicine, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110.
Jonathan
of Laboratory
63110
and
11The
H. Hughes+,
Medicine Upjohn
John
October 23, 1989) Turk+**,
and
and Department of Internal Medicine, Company, Kalamazoo, Michigan 49001
to isolated islets (9-11). Phorbol ester-induced insulin secretion is temporally associated with an increase in membraneassociated protein kinase C activity (7, 12) and is suppressed by pharmacologic inhibitors of protein kinase C (7, 13, 14). Glucose-induced insulin secretion from rat islets is also suppressed (60-100%) by such inhibitors suggesting a role of protein kinase C in this process (7,12,13). In contrast, glucose does not induce the translocation of protein kinase C activity to a membrane-associated state (7, 12). Furthermore, islets from which protein kinase C activity has been quantitatively depleted by prolonged (20-24 h) incubation with phorbol esters secrete insulin normally in response to glucose, although they no longer secrete insulin in response to further stimulation by phorbol ester (2,15). The role of protein kinase C activation in glucose-induced insulin secretion is therefore controversial. In a variety of cell types, the activation of protein kinase C is accompanied by the increased phosphorylation of an 80kDa protein which is a widely distributed endogenous substrate for the enzyme (16, for reviews see Refs. 17-19). Recently, Stump0 et al. (20) have isolated and sequenced a cDNA clone encoding the gene for the bovine 80-kDa protein kinase C substrate and have determined that the molecular mass of this protein is in fact 31.9 kDa. This protein and the equivalent protein purified from fibroblasts were shown to be myristoylated and to contain a high proportion of alanine and acidic amino acid residues (21-23), and the 80-kDa protein has been designated the MARCKS’ protein (20). This protein is rapidly phosphorylated following the activation of protein kinase C by such stimuli as growth factors (24, 25) and bombesin (26) in fibroblasts and by neurotransmitters in neuronal cells (27). It is also phosphorylated in numerous cell types in response to activation by phorbol esters and cellpermeable diacylglycerols (16,18,24-27). It is not phosphorylated, however, in cells that are stimulated with agonists, e.g. prostaglandin E1 and epidermal growth factor, that do not activate protein kinase C (18, 28, 29). The 80-kDa protein is therefore considered to be a selective endogenous substrate for protein kinase C whose phosphorylation state specifically reflects the activation of this enzyme in intact cells (17, 25, 30). ’ The abbreviations used are: MARCKS, myristoylated alaninerich C kinase substrate: TPA, 12-0-tetradecanoylphorbol 13-acetate; EGTA, [ethylenebis(ox>ethyienenitrilo)]tetraacetic acid; TES, 2-([2hydroxyl-l,l-bis(hydroxymethyl)ethyl]-amino~ethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; Me,SO, dimethyl sulfoxide; PDBu, phorhol 12,13-dibutyrate; ANOVA, analysis of variance.
14938
Protein In an attempt glucose-induced
to clarify the insulin secretion,
Kinase C and Insulin
role of protein kinase the phosphorylation
C in of the
80-kDa protein has been utilized as an indicator of the activation of protein kinase C in intact islets. This experimental approach has been used to compare the effects of three distinct insulin secretagogues on phosphorylation of the 80-kDa protein with their effects on insulin secretion. The compounds studied are the fuel secretagogue D-glucose, the muscarinic agonist carbamylcholine (carbachol), and the phorbol ester 12-O-tetradecanoylphorbol13-acetate (TPA). EXPERIMENTAL
PROCEDURES
Materials-Male Sprague-Dawley rats (160-180 g) were purchased from Sasco (O’Fallon, MO) and maintained on Rodent Chow 5001 (Ralston Purina, St. Louis, MO) ad Zibitum for at least 7 days prior to use. The sources of materials are as previously described (7). Normal rabbit serum was obtained from Sigma. Pansorbin (Cells) was obtained from Calbiochem (San Diego, CA). Antibody raised to an amino-terminal peptide of the MARCKS protein was a generous gift of Dr. Perry Blackshear. In addition, “*Pi was purchased from New England Nuclear Research Products, and [“Hlphorbol 12,13dibutyrate was obtained from Amersham Corp. Isolation and Culture of Islets-Pancreatic islets were isolated aseptically from male rats as previously described (31). Islets were cultured overnight at 24 “C in tissue culture medium CMRL-1066 containing 5.5 mM glucose, 1% L-glutamine, 10% heat-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin under an atmosphere of 95% air, 5% CO,. Preparation of a Heat-stable Protein Fraction from Isolated ZsletsIslets obtained from six to eight rats were homogenized by 13 passes in a Teflon/glass homogenizer in buffer containing 10 mM Tris-HCl, pH 7.5, 250 mM sucrose, 10 mM dithiothreitol, and 2 mM EGTA. A heat-stable protein preparation enriched in the 80-kDa protein kinase C substrate was prepared from this extract by a method previously described by Blackshear et al. (32). Phosphorylution of Islet 80.kDa Protein in Vitro-Islets obtained from six rats (-1800 islets) were disrupted by 13 passes of a Teflon/ glass homogenizer in 400 ~1 of ice-cold buffer containing 25 mM TESNaOH, pH 7.4, 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 250 rg/ml bovine serum albumin (BSA), and 100 pg/ml leupeptin. The homogenate was centrifuged (Beckman SW50.1 rotor, 135,000 x g, 60 min, 4 “C) and the supernatant retained as a source of protein kinase C. In vitro phosphorylation of islet cytosolic proteins was performed in a total volume of 100 ~1 of assay medium containing 100 mM TESNaOH, pH 7.4, 5 mM MgCl,, 1 mM EGTA & 1.3 mM Ca” (free Ca” concentration -300 PM), +125 pg/ml phosphatidylserine, and f2.4 pg/ml 1,2-diolein. To each tube was added 20 J of the heat-stable protein preparation (-1-3 eg of protein) and 10 ~1 of islet cytosol extract. After a preincuhation period of 2 min, the reaction was initiated by the addition of [r-“‘P]ATP (10 +l, final concentration, The incubation was terminated at 1 min 20 PM, -30-40 &i/tube). by the addition of 25 ~1 of 50% trichloracetic acid (final concentration lo-15%, w/v). The tubes were then vortexed and placed on ice. After 30 min, the samples were centrifuged (Beckman microfuge 12,000 x g, 5 min) and the supernatant aspirated. The tubes were then washed with ice-cold acetone (1 ml) and the tubes allowed to air dry. The samples were then prepared for two-dimensional SDS-PAGE electrophoresis as described below. Phosphorylation of the Islet BO-kDa Protein in Situ-For phosphorylation studies, isolated islets were washed three times in KrebsRinger-Hepes buffer (25 mM Hepes, pH 7.4, 115 mM NaCl, 24 mM NaHC03, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgC12) containing 3 mM glucose, and 0.1% BSA (basal medium). One-hundred (100) islets were individually selected under a stereomicroscope and placed in polyallomer tubes (Beckman, Palo Alto, CA). The bathing medium was then removed with a drawn-out Pasteur pipette and replaced with basal medium (200 ~1) containing “‘Pi (200 PCi). The islets were then preincubated for 60 min in a water bath at 37 “C with gentle shaking to label endogenous ATP pools. Incubations (1, 5, 10, or 30 min) were initiated by addition of a concentrated (40-fold the final concentration) solution (5 ~1) of secretagogue (carbachol, glucose, or TPA). The incubation mixture was gently mixed in the tubes using a stream of gas (95% air, 5% CO,), and the tubes were then returned to the water bath at 37 “C.
Secretion
14939
The incubation was terminated by centrifugation (Beckman Microfuge 12,000 x g, 5 s), aspiration of the medium with a drawn-out Pasteur pipette, and freezing (dry-ice) the pelleted islets. Preparation of Samples for Two-dimensional SDS-PAGE-The sedimented islets were resuspended in ice-cold buffer (150 ~1) containing 50 mM MES, 1 mM EDTA, 250 mM sucrose, pH 7.2, and homogenized by sonication (Branson Sonifier 350,5 s, setting 3). Cell debris was then removed by centrifugation (Beckman Microfuge 12,000 x g, 2 min, 4 “C) and trichloroacetic acid added to the supernatant (final concentration lo%, w/v). The samples were chilled on ice (30 min) and the cytosolic proteins were then collected by centrifugation (Beckman microfuge 12,000 X g, 2 min). The tubes were washed with 2 X 1.0 ml of ice-cold acetone and allowed to air dry. In several experiments, inhibitors of protein phosphatases (i.e. 100 mM NaF and/or 50 mM benzamidine) were included in the processing buffer to eliminate the possibility that the phosphorylation of islet 80-kDa protein was rapidly reversed during the processing procedure. These agents, particularly NaF, were found to interfere with sample recovery as indicated by reduction in radioactivity associated with protein spots after electrophoresis. Since experiments including these treatments did not indicate that significant dephosphorylation was occurring, subsequent experiments were performed in the absence of these inhibitors. Two-dimensional SDS-PAGE-Samples were solubilized in 9 M urea and separated by two-dimensional polyacrylamide gel electrophoresis by the method of Anderson and Anderson (33). Isoelectric focusing was performed in the first dimension in a 50:50 mixture of Ampholyte 3/10 Bio-Lyte (Bio-Rad) and Servalyt 3-10 (Serva, Westbury, NY). SDS-polyacrylamide gel electrophoresis was conducted in the second dimension using 10% gels. After electrophoresis, the gels were dried, exposed to x-ray film, and the area of the gel in which the phosphorylated 80-kDa protein kinase C substrate was located and scanned with a Beckman DU-8 spectrophotometer with a gel scanning attachment. Data are reported as the area under the resulting tracing. Zmmunoprecipitation of MARCKS Protein from Islets-Islets (175/ tube) were incubated in the presence of 200 &i of 32Pi for 60-75 min at 37 “C as described above. The islets were then incubated with either TPA (200 nM) or vehicle (0.1% MeSO) for 10 min at 37 “C. Following this incubation, the islets were sedimented by brief centrifugation (12,000 x g, 5 s) and the supernatant removed. The islets were resuspended in ice-cold buffer (400 pl, 50 mM Tris-HCl, pH 8.3, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40) and sonicated (5 s, 4 “C, setting 2-3, Branson Cell Disruptor, Danbury, CT). The homogenate was placed on ice for 15-30 min and then clarified by ultracentrifugation (135,000 x g, 30 min, 4 “C). A portion (5 ~1) was added to a y-globulins solution (400 ~1 of a 1 mg/ml), and the radioactive phosphate incorporated into protein was determined after trichloroacetic acid (25% w/v) precipitation as described above. The remaining sample was precleared by a procedure which involved the addition of normal rabbit serum (4 ~1/400 ~1 sample) and subsequent incubation at 4 “C for 30-60 min with gentle rotation. To each sample was then added 50 ~1 of Pansorbin Cells (10% w/v) which had been washed (at least three times) with wash buffer (50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40). After a further incubation (30-60 min) at 4 “C with rotation, the Pansorbin was precipitated by centrifugation (12,000 x g, 5 min, 4 “C). The samples were then incubated with primary antibody (5 bl/sample) or normal rabbit serum (as control) and incubated (with rotation) for a further 30-60 min at 4 “C. Following this incubation, the primary antibody was immunoprecipitated by Pansorbin as described above. The resulting pellet was washed four times with 1 ml of wash buffer, solubilized in 9 M urea, and subjected to two-dimensional SDS-PAGE electrophoresis. PHIPhorbol Ester Binding to Subcellular Fractions of Islets-Islets obtained from 15 rats (-4500) were washed in basal medium and distributed by counting into three siliconized scintillation vials (lOOO1500 islets/vial). The islets were then preincubated in 1 ml of basal medium containing 3 mM glucose for 30 min in a 37 “C water bath with gentle shaking. This medium was then gently aspirated and replaced with basal medium with or without carbachol (500 NM) or glucose (16.5 mM). After 5 min, this medium was aspirated, and its insulin content was subsequently measured by radioimmunoassay. The remaining islets were disrupted by 13 passes of a Teflon/glass homogenizer in ice-cold buffer (400 ~1) containing 25 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2.5 mM EGTA, 2.5 mM Mg acetate, 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 100 rg/ml leupeptin. The homogenate was centrifuged (Beckman SW50.1, 135,000 X g) for 1 h at 4 “C. The resulting supernatant was retained
14940
Protein Kinase C and Insulin
for the determination of specific binding activity for the radiolabeled ligand [“H]phorbol 12,13-dibutyrate (PDBu) by cytosolic protein kinase (7. The pellet was resuspended in homogenization buffer (300400 ~1) and used for the determination of binding activity by membrane-associated protein kinase C. Specific [“H]PDBu binding to these subcellular fractions (20-50 ~1) was determined as described by daken (34). A concentration of [“H]PDBu between ‘LO-40 nM was employed and nonspecific binding was measured in the presence of 100 PM unlabeled PDBu. The binding assay was performed at 4 “C for 1 h.
Insulin
Secrc~liofz-In
counted (20 or “I.i/tube) tubes and preincuhated basal medium containing
static secretion experiments,
Secretion
(PH)
of
Islet 80-kDa
Ca’+ and Phospholipid
Protein Is Stimulated in Vitro
by
been used as an intracellular
80-kDa protein has marker of the activation of
protein
cell types. The initial
The phosphorylation
kinase C in numerous
,
68 -46 -36 -27
ca”Psw 3
+ca2+PsBG , 8
I
C + ,*
I -
INTACT ISLETS
9
? c
-27 ‘y.
b-
.
--PA
FIG. 1. Protein kinase C-mediated phosphorylation of an 80-kDa protein by islet cytosol in vitro and in intact islets in situ. Two-dimensional SDS-PAGE of samoles oreoared as described under “Experimental Procedures” was performed, and autoradiographs are shown here. Quadrants A and E are samples of islet cytosol extract which had been incubated in uitro in the absence (A) or presence (B) of Cal+, phosphatidylserine (PS), and 1,2-diolein (DAG). Quadrants C and D are samples of 100 islets which had been incubated in situ for 15 min in the presence of carrier alone (0.1% MeLSO) (C) or 100 nM TPA (D). The position of the 80-kDa protein in both conditions is indicated by the arrow. The position of a 54-57kDa protein(s) visible in the in vitro incubation using islet cytosol is indicated by the arrowhead (see Table I). The pH range used in isoelectrofocusing gels was 7.5 (at the right-hand side of the gel as represented) to 3.5 on the left (as shown above panels A and B). The positions of the molecular mass markers (kDa X 10-l) run on the same gels are shown on the right. I
L .
activation
of Ca’+-calmodulin-dependent
that was observed
protein
TABLE
may
kinase
(38)
and tubulin (39), and their phosphorylation is known to be stimulated by Ca’+ and calmodulin. Since the islet cytosol
I
Specific phosphorylation of an 80-kDa protein by protein kinase C in vitro
aim of
reflect the presence of small amounts of phospholipids in the cytosol fraction prepared from islets. In contrast, addition of Cay+ alone did promote, to a significant degree, the phosphorylation of a protein(s) of molecular mass 54-57 kDa (Fig. lB, Table I). Proteins of this molecular weight include the subunits
-116 a4
of an endogenous
the present study was to determine whether the phosphorylation of a similar 80-kDa protein reflected the activation of protein kinase C in islets. Fig. 1 (A and B) illustrates an autoradiogram of two-dimensional SDS-PAGE analysis of proteins phosphorylated by a cell-free extract of islet cytosol. This figure demonstrates that an acidic protein of M, - 80 kDa was phosphorylated in response to the activation of protein kinase C in islet cytosol by the addition of the cofactors of protein kinase C (Ca”, phosphatidylserine, and diacylglycerol). The isoelectric point of this phosphorylated protein was 4.0-4.5 pH units and thus was very similar to that reported for the 80-kDa protein kinase C substrate in other cell types (16, 24-26). Quantitative analysis of autoradiograms of these in vitro experiments demonstrated that the activation of protein kinase C by the simultaneous addition of Ca’+, phosphatidylserine, and diacylglycerol resulted in a g-fold increase in 80kDa protein phosphorylation (Table I). Phosphorylation of the islet 80-kDa protein by rat islet cytosol was not significantly influenced by Ca’+ alone (free concentration -300 jiM) The modest, nonsignificant
75
.’
CMOSOL
air/CO, (9.5/S%). The medium was replaced with fresh basal medium
Phosphorylation
6s B
/
into 12 X 75-m, siliconized borosilicate for 30 min at 37 “C with gentle shaking in 0.1% BSA (200 ~1) under an atmosphere of
RESULTS
35
A
islets were
alone or supplemented with glucose (10 mM), carbachol (500 PM), or a combination of glucose and carbachol. The incubation was then continued for a further 30 min and was terminated by the removal of medium. Insulin content of this supernatant medium was determined by radioimmunoassay. In protein kinase C inhibition experiments, staurosporine (100 nM) was present during both the preincubation and incubation periods. In the perifusion experiments, islets were placed in chambers (100 islets/chamber) and perifused with basal medium containing 0.1% BSA for 30 min at 37 “C as previously described (35, 36). During the last 5 min of this preincubation period staurosporine (final concentration 100 nM) or vehicle (0.1% MeaSO) was added. The chambers were then perifused with the same medium supplemented with glucose (10 mM) and carbachol (500 PM) and containing either vehicle or staurosporine (100 nM) for a further 40 min. The insulin content of the perifusion effluent was measured by radioimmunoassay. Protein Estimation-Protein concentration was estimated by t.he method of Bradford et al. (37) using bovine serum albumin as standard.
7.5
55
2.5
Islet cytosol was incubated in the presence of EGTA (1 mM), Ca” (-300 pM) alone or Ca” (-300 pM) in the presence of phosphatidylserine (PS, 125 ,ug/ml) and 1,2-diolein (DAG, 2.4 pg/ml). The assay also included 1.3 pg of a heat-stable protein fraction prepared from islets. The samples were analyzed by two-dimensional SDS-PAGE and ‘“Pi incorporation into the 80-kDa protein and the 54-57-kDa protein(s), indicated by the arrowhead (see Fig. lB), quantitated by laser densitometry. Data represent four separate experiments which all yielded similar results. P, incorporation Additions 54-57 kDa SO kDa
arbitrary units None Ca?’
Ca” > PS > DAG
0.69 + 0.05 1.41 + 0.41 6.40 + 0.87
0.20 + 0.04 5.78 + 0.33 3.33 + 0.37
preparation used in these assays contains a high concentration of calmodulin, the phosphorylation of this 54-57-kDa protein(s) is likely due to the action of a Ca’+-calmodulindependent protein kinase. The data in Table I, therefore, demonstrate that the 80-kDa protein in islets is selectively phosphorylated by the activation of protein kinase C in vitro and that its phosphorylation state is not increased under conditions in which the Cap+-calmodulin-dependent protein kinase is activated. As demonstrated in Table II, protein kinase C-mediated phosphorylation of the islet 80-kDa protein was suppressed by the inclusion of the protein kinase C inhibitors, staurosporine (7, 40) and K252b (41). Staurosporine, at a concen-
Protein
Kinase C and Insulin
TABLE II of 80.kDa
Prevention
protein phosphorylation by inhibitors of protein kinase C
islet protein is in fact the MARCKS protein previously described from other tissues and validates its use as an indicator of the activation of islet protein kinase C.
in vitro
Islet cytosol was incubated in the absence and presence of Cay+, phosphatidylserine (PS), and diolein (DAG) (as described in the legend to Table I) and in the absence and presence of the protein kinase C inhibitors K252b and staurosporine. The samples were analyzed by two-dimensional SDS-PAGE and the “PI incorporation into the islet 80-kDa protein quantitated by densitometry. Additions Inhibitor CIXIC. P, incorporation arbitrary
None +Ca”+ +Ca’+’ +Ca’+: +Ca’+,
PS PS’ PSI PS,
DAG DAG DAG DAG
units
0.19 + 0.08 2.82 + 0.25
K252b K252b Staurosoorine
1001WnM
50 nM
0.16 0.03 0.02
+ 0.13 0.02
+ 0.01
tration of 50 nM totally prevented this phosphorylation. In other experiments, staurosporine at a concentration of 10 nM inhibited this phosphorylation by >95%. K252b is an inhibitor which shows considerable specificity toward protein kinase C relative to other kinases (41). At concentrations of K252b which inhibit islet protein kinase C activity (assayed by histone phosphorylation) by >95% (data not shown), this compound also completely prevented the phosphorylation of the 80-kDa protein by islet cytosol. Phosphorylation
of the 80-kDa Protein Is Stimulated Phorbol Esters in Intact Islets
by
The phorbol ester TPA is a potent activator of protein kinase C, and therefore its ability to increase the phosphorylation site of the 80-kDa protein in intact islets in situ was assessed. As shown in Fig. 1 (C and D), the “Pi incorporated into an acidic protein of M, - 80 kDa (indicated by the arrow) was significantly increased in islets incubated in the presence of TPA. This phosphoprotein found in intact islets corresponded on two-dimensional SDS-PAGE to the protein phosphorylated by the activation of protein kinase C in vitro with respect to isoelectric point, size, and shape (cf. Fig. 1B). The incubation of islets (15 min) with TPA (100 nM or 1 PM) increased the phosphorylation of islet 80-kDa protein (by 222 and 320%, respectively, as expressed as percentage of the control at 3 mM glucose). Both increases were statistically significant (p = 0.007 and 0.015, respectively). Phosphorylation of the 80-kDa protein stimulated by TPA (100 nM) was significantly suppressed (-75%) by staurosporine (100 nM; 125 f 22% versus 199 f 44%, expressed as a percentage of basal). Phosphorylation of the 80-kDa protein was clearly the most prominent TPA-sensitive phosphorylation. Although other TPA-sensitive phosphorylations were observed (see Fig. 1) they were both less intense and less consistent. Immunoprecipitation of Islet 80-kDa Raised to the MARCKS
Protein Protein
14941
Secretion
Secretagogue-induced 80-kDa Protein
Phosphorylation Kinase C Substrate
of the Endogenous in Intact Islets
Effects of Carbachol-Carbachol is known to promote cellular responses by muscarininc receptor-mediated activation of phospholipase C and the subsequent production of inositol phosphates (including inositol trisphosphate (42) and diacylglycerol (11, 52). Inositol 1,4,5-trisphosphate is known to stimulate the release of Ca*+ from the islet endoplasmic reticulum (35) and thus to increase the intracellular Ca’+ concentration (43). It is well established that diacylglycerol is an endogenous activator of protein kinase C (44). Carbachol would therefore be expected to promote phosphorylation of the endogenous 80-kDa protein kinase C substrate in islets. As demonstrated in Fig. 3, carbachol (500 pM) did in fact induce a marked increase in the RZPi incorporation into the islet 80-kDa protein. This effect was transient, with maximal incorporation at 5 min (219 f 22% of control, p = 0.015). By 10 min, the “Pl incorporation had decreased to 157 + 19% of control 0, = 0.005). Under similar conditions, carbachol (500 pM) induced approximately a 2.6-fold increase in insulin secretion over a 5-min incubation period (Table II). Carbachol-induced phosphorylation of the islet endogenous 80kDa protein kinase C substrate was suppressed in a dosedependent manner by the protein kinase C inhibitor, staurosporine (Fig. 4). Carbachol-induced insulin secretion was also partially suppressed (-42%) by staurosporine (100 nM). These data indicate that carbachol induces the time-dependent activation of protein kinase C in intact islets and suggest that this mechanism may participate in the stimulation of insulin secretion by this muscarinic agonist. Effect of Glucose-As shown in Fig. 3, a concentration of glucose (28 mM) that maximally stimulates insulin secretion from isolated islets also promoted phosphorylation of the islet endogenous 80-kDa protein kinase C substrate. Glucose-induced incorporation of “*Pi into this 80-kDa protein was maximal at 5 min (128 + 20% of control, p = 0.25) and was reduced to 111 + 12% of control by 10 min. This time course mimicked that observed with carbachol, but the effect of glucose was small and did not achieve statistical significance as assessed by two-way analysis of variance (ANOVA) (Fig. 2). It has been proposed (45) that protein kinase C preferentially regulates the second phase of insulin secretion. The effect of glucose at later time points was therefore examined. Glucose (28 mM) was not found to induce a significant increase in the phosphorylation of the 80-kDa protein in islets after a 30-min incubation (116 f 16% of control), although second phase insulin secretion is well established at this time.
by Antibody
In order to confirm the identity of the islet 80-kDa protein as the MARCKS protein, an antibody raised to amino-terminal peptide of the MARCKS protein was obtained from Dr. Perry Blackshear. This antibody was used to immunoprecipitate phosphoproteins from islet homogenates previously labeled with ‘lP,. As illustrated in Fig. 2, this antibody immunoprecipitated a phosphoprotein equivalent to the islet 80kDa protein phosphorylated by protein kinase C with respect to molecular mass, acidic isoelectric point, and distinctive shape (cf. Fig. 1). Furthermore, the phosphorylation of this protein was also increased by treating islets with TPA (Fig. 2B cf. Fig. 2A). This protein was not precipitated by normal rabbit serum. These observations indicate that the 80-kDa
Secretagogue-induced
Translocation
of Protein
Kinase
C
To confirm the results of the phosphorylation studies, another approach was also used to examine the influence both of carbachol and of glucose on protein kinase C activation in islets. This approach involved the assessment of the effect of these insulin secretagogues on the translocation of protein kinase C to a membrane-associated state. Since different protein kinase C isoenzymes may exist in islet P-cells (46) and may have distinct functions and/or different substrate specificities (471, a phorbol ester binding procedure was used to detect the mass of protein kinase C associated with cytosolic and particulate (membranous) fractions of islets stimulated with carbachol or glucose. The radiolabeled phorbol ester [“H]PDBu was found to bind either to cytosolic or to
14942
Protein
Kinase
C and Insulin
A
Secretion B
-160 .116
FIN. 2. Immunoprecipitation of islet 80-kDa protein by MARCKS protein antibody. Immunoprecipita-
>.
J
-84
tion was performed on islets incubated in the presence of vehicle (0.1% Me$O) (panel A) of 200 nM TPA (panel H) as described under “Experimental Procedures.” Shown above are autoradiograms of’two-dimensional SDS-PAGE analyses performed on these samples under conditions identical to those described in the legend to Fig. 1.
-36
-27
40
5.5 - TPA
+TPA
Synergy .. .
I
0
2
4
I
6
8
10
TIME (min)
FK
3. CarbacholSO-kDa protein
and glucose-induced phosphorylation in islets. Islets (loo/tube) were labeled
of
with lLP, as described under “Experimental Procedures.” The islets were then incubated in the presence of carbachol (500 pM W) or glucose (28 mM 0) for the time indicated. The samples were analyzed by twodimensional SDS-PAGE and the “‘P, incorporated into the 80-kDa protein quantitated by laser densitometry. The data is expressed as a percentage of P, incorporated in the presence of 3 mM glucose (basal conditions). *p < 0.02, **p < 0.002, ***p < 0.0001 uersus control, twoway analysis of variance (ANOVA). The effect of carbachol as compared to glucose was also significant at 1 min (p = 0.029) and 5 min @ = 0.0005). the
particulate protein kinase C with a Kd of 0.5-2.0 nM (not shown). Carbachol (500 PM), under conditions which promoted a 2.6-fold stimulation of insulin secretion, induced a significant increase in the specific binding of [“H]PDBu by the membranous (particulate) fraction of islets compared to basal conditions (Table III). By contrast, stimulatory concentrations of glucose did not increase the mass of protein kinase C associated with the membrane fraction of islets despite inducing a 3-fold increase in insulin secretion.
5.5
between Glucose and Carbachol on Insulin Secretion and Phosphorylation of the 80-kDa Protein
It is known that the insulin secretagogue effect of carbachol requires the presence of at least basal glucose concentrations (3-5 mM) and is potentiated in the presence of submaximal concentrations of glucose (e.g. 10 mM) (1). This observation has been confirmed in the present study (Fig. 5A). Glucose (10 mM) or carbachol (500 PM) induced a 6- or 3-fold increase in insulin secretion, respectively, as expressed as a percentage of control (Fig. 5A). The simultaneous addition of these secretagogues promoted a 30-fold increase in insulin secretion compared with basal glucose concentrations (3 mM) over a 30-min period. It was of interest, therefore, to examine whether glucose influenced the carbachol-induced phosphorylation of the 80-kDa protein. As shown in Fig. 5B, and consistent with data shown in Fig. 3, glucose (10 mM) alone did not increase the phosphorylation state of the 80-kDa protein under conditions in which carbachol (500 PM) promoted a 1.74-fold increase compared with islets that had been incubated in basal medium (p = 0.0097). The simultaneous addition of glucose (10 mM) and carbachol (500 PM) did, however, significantly increase the phosphorylation (277 + 35%) of the 80-kDa protein either compared with basal phosphorylation levels (p < 0.0001, ANOVA) or compared with phosphorylation induced by carbachol alone @ = 0.0007, ANOVA). Experiments performed with the phorbol ester binding assay indicated that the simultaneous addition of glucose (10 mM) and carbachol (500 pM) did not significantly increase [:‘H]PDBu binding by membranous fractions over that induced by carbachol alone (data not shown). The hypothesis that the increased activation of protein kinase C might mediate the synergistic insulin secretory response to the addition of the combination of glucose and carbachol was tested by examining whether staurosporine was able to suppress insulin secretion under these conditions. As shown in Fig. 6B, insulin secretion was inhibited in a dosedependent manner by staurosporine. Maximal inhibition (-31%) was achieved at 100 nM staurosporine. In islet peri-
Protein Kinase C and Insulin
260
14943
Secretion
q
: Vehicle
1
: 100nM Staurosporine
B
-
FIG. 4. Effect of staurosporine on carbachol-induced phosphorylation of islet 80-kDa protein (A) and insulin secretion (B). A, islets (loo/tube) were labeled with 32Pi and incubated with basal medium alone (in the presence of 0.1% MeSO), or basal medium supplemented with carbachol (CCh) (500 pM, 0.1% MeSO) or carbachol in the presence of staurosporine (l-100 nM). The samples were analyzed by two-dimensional SDSPAGE and the “‘Pi incorporated into the 80-kDa protein quantitated by laser densitometry. B, islets (25/tube) were preincubated in basal medium containing vehicle (0.1% Me2SO) or staurosporine (100 nM) for 30 min at 37 “C. The islets were then incubated with basal medium or basal medium containing carbachol (500 PM) in the absence and presence of staurosporine (100 nM). The data is expressed as a delta (A) of insulin secretion induced by carbachol less that secreted in basal medium. Staurosporine had no effect on basal insulin secretion. TABLE
III
[‘H/PDBu
binding to cytosol and particulate protein kinase C in islets stimulated with glucose or carbachol Islets (1000-1500) were incubated with glucose or carbachol for 5 min. Following the removal of the incubation medium, the islets were homogenized and cytosol and particulate fractions obtained by ultracentrifugation. [3H]PDBu binding to these fractions was determined as described under “Experimental Procedures.” The insulin content of the incubation medium was determined by radioimmunoassav. Condition
[“H]PDBu
NO.
bound
Particulate
Cvtosol
fmol/islet
3 mM
glucose 16.5 mM glucose 3 mM &COW + 500 pM car-
6
0.595
+ 0.051
0.137
5
0.530
6
0.498
-+ 0.043 + 0.049
0.138 0.199
Insulin
secretion punits/islet/5
+ 0.019 + 0.039 f 0.016*
min
2.13 f 0.40 6.56 f
1.71
5.61 f
1.70
bachol * p < 0.05 versus
3 mM glucose.
staurosporine was found not to of secretion (Fig. 6A). In some experiments staurosporine actually potentiated first phase secretion slightly. In contrast, staurosporine profoundly suppressed second phase secretion (Fig. 6A). This selective inhibition of second phase insulin secretion by staurosporine likely explains the observation that staurosporine only partially inhibits insulin secretion in a static secretion model carried out over a period of 30 min. Both first and second phase secretion contribute to the total insulin secreted in such static experiments. Since staurosporine inhibits only second phase, and not first phase insulin secretion, total insulin secretion is only partially inhibited by maximally effective concentrations of staurosporine in static experiments. This phenomenon also suggests that protein kinase C may play an important role in the regulation of second phase insulin secretion. fusion secretion influence the
experiments first phase
DISCUSSION
Insulin secretion from islets can be stimulated by secretagogues of at least two general classes. The muscarinic neurotransmitter acetylcholine, induces insulin secretion by inter-
action with P-cell plasma membrane receptors and is thought to be involved in the cephalic phase of insulin secretion (48). D-Glucose, the major physiological insulin secretagogue, is an example of a “fuel secretagogue,” so-called because its metabolism is required to induce insulin secretion (1). The present study has assessed the role of protein kinase C in these mechanisms by comparing the effects of these secretagogues on the phosphorylation of the endogenous 80-kDa protein kinase C substrate in islets. The phosphorylation of this endogenous 80-kDa protein has been used as an indicator of the activation of protein kinase C in numerous cell types in response to a variety of agonists. The advantage of this approach is that an assessment of protein kinase C activity can be obtained in intact cells. This reduces the possibility of artifacts which have been suggested to occur in disrupted cell assays (7, 17). The use of the phosphorylation of the 80-kDa protein in islets as an indicator of the activation of protein kinase C was validated in several different ways. 1) It was demonstrated that the islet 80-kDa protein was phosphorylated in vitro under conditions which are known to activate protein kinase C, i.e. in the (see Ref. 7). In addition, presence of Ca2+ and phospholipid this phosphorylating activity could be totally eliminated by the inclusion of the protein kinase C inhibitors staurosporine and K252b. 2) Second, the islet 80-kDa protein was phosphorylated in intact islets in situ in response to TPA, which activates protein kinase C in islets (7, 12). This event was also prevented by staurosporine. 3) Immunoprecipitation studies using the anti-MARCKS antibody were performed to demonstrate that the islet 80-kDa protein is closely related to or identical to the MARCKS protein. This protein has been shown to be a selective substrate for protein kinase C (24-25, 28, 30). The cellular function of this protein is not yet established but recent studies have demonstrated that it is myristoylated and has the capacity to bind and release calmodulin in the non-phosphorylated and phosphorylated forms, respectively (49). These observations suggest that the MARCKS protein may have an important role in the regulation of acute cellular responses (50). Carbachol was found to induce the phosphorylation of this
14944
Protein Kinase C and Insulin
Secretion
1cwdgkms6z cE= %F+=‘WCCh
% low= +Wm
FIG. 5. Synergistic effect of glucose and carbachol on insulin secretion (A) and 80-kDa protein phosphorylation (B) in islets. A, islets (2O/tube) were incubated for 30 min at 37 “C in the presence of glucose, carbachol (CCh), or both (see “Experimental Procedures”). The insulin content of the incubation medium was determined by radioimmunoassay. B, the phosphorylation experiments were performed as described in the legend to Fig. 2. Prelabeled islets were incubated with secretagogues for 5 min. *p = 0.0097 versus control, **p = 0.0007 uersus carbachol. ANOVA.
3mM glucose
1OmM glucose
+I-100nMstaumspline
A
60
+ 5rnpM carbach
B II
4
70
3 60 2 50 1
0
40 0
20
40
60
TIME (mln)
0
20
40
60
STAUROSPORINE
60
100 (nM)
FIG. 6. Effect of staurosporine on insulin secretion induced by the simultaneous addition of glucose and carbachol. A, islet perifusion. Islets (lOO/‘chamber) were perifused at 37 “C with basal medium for 25 min followed by basal medium supplemented with MezSO (O.l%, open symbols) or staurosporine (100 nM, closed symbols) for 5 min. Islets were then perifused for a further 40 min with medium containing glucose (10 mM) and carbachol (500 FM) supplemented with vehicle or staurosporine (100 nM). B, static secretion. Islets (20/tube) were preincubated in conditions described in the legend to Fig. 4. The islets were then incubated with medium containing glucose (10 mM) and carbachol (500 pM) supplemented with vehicle (0.1% Me$O) or staurosporine (3-100 nM). After 30 min the medium was withdrawn and the insulin content determined by radioimmunoassay.
80-kDa protein in islets in a time-dependent manner reflecting the activation of protein kinase C. This result is consistent with the prevailing view that the cellular responses of this muscarinic agonist are mediated by receptor-coupled stimulation of phospholipase C-catalyzed hydrolysis of membrane phospholipids (1). Carbachol has been shown to induce the formation of inositol phosphates (including inositol trisphosphate) in islets (42, 51, 52). More recently, it has been shown that carbachol induces the accumulation of [3H]diacylglycerol from [3H]glycerol labeled islets and that the fatty acid composition of this diacylglycerol is consistent with its derivation in part from membrane phosphoinositides (9,53). It is striking that the time course of carbachol-induced phosphorylation of the islet 80-kDa protein corresponds very closely to the time course of carbachol-induced accumulation of [3H]diacylglycerol in islets prelabeled with [3H]glycerol (9). These observations support the interpretation that carbachol-induced phosphorylation of the islet 80-kDa protein is mediated by the activation of protein kinase C induced by the accumulation of endogenous diacylglycerol. The data from the [3H]PDBu binding assays indicate that carbachol-induced activation of islet protein kinase C is accompanied by its translocation to a membrane-associated state and agree with preliminary observations
(11,
54).
kDa phosphorylation
Furthermore,
the
and the 2.6-fold
2.2-fold
increase
stimulation
in 80-
of insulin
secretion in carbachol-stimulated islets are quantitatively similar. Finally, the temporal correspondence of diacylglycerol accumulation and insulin secretion observed previously (9) in carbachol-stimulated islets suggest a role for protein kinase C in carbachol-induced insulin secretion. The fact that staurosporine only partially inhibits carbachol-induced insulin secretion suggests that the activation of protein kinase C is not the sole mechanism mediating the insulin secretory response of islets to this agonist. By contrast, stimulatory concentrations of glucose alone did not induce a significant increase in the phosphorylation of the endogenous islet 80-kDa protein kinase C substrate. This indicates that glucose alone does not strongly activate protein kinase C in islets. This interpretation is supported by the observation that glucose, despite inducing an insulin secretory response even more robust than that induced by carbachol, did not induce the translocation of protein kinase C mass to an activated, membrane-associated state as assessed by [3H]PDBu binding (Table III). These observations are consistent with previous reports the glucose does not induce the translocation of protein kinase C activity, as measured by a histone phosphorylation assay (7, 12). Furthermore, it has recently been shown that glucose-stimulated islets do not accumulate an increased mass of diacylglycerol as determined by a diacylglycerol kinase assay (11). The sum of these accu-
Protein Kinase C and Insulin mulated observations indicates that glucose-induced insulin secretion is mediated principally by mechanisms independent of the activation of protein kinase C. The combination of carbachol and a submaximally effective concentration of glucose (10 mM) was, however, found to act synergistically to increase the phosphorylation of the endogenous 80-kDa protein kinase C substrate in islets, and there was a corresponding synergistic effect of the combination of carbachol and glucose to increase insulin secretion. These observations suggest that the synergistic effect of these secretagogues on insulin secretion might be mediated by the activation of protein kinase C. Consistent with this hypothesis, insulin secretion induced by the combination of these two secretagogues was found to be suppressed significantly by the protein kinase C inhibitor staurosporine. This inhibitory effect of staurosporine was observed only on the second phase and not the first phase of insulin secretion. These observations provide further evidence that the activation of protein kinase C may amplify second phase secretion. That synergistic activation of protein kinase C was not observed in the [3H] PDBu binding translocation assay may reflect direct activation of membrane-associated protein kinase C by glucose or synergistic stimulation of diacylglycerol accumulation which competes with the labeled phorbol ester for binding. Amplification of carbachol-induced insulin secretion by glucose is well documented (55,56). This may result from the ability of glucose to promote the entry of Caz+ by depolarization of the P-cell (1, 56, 57). There is evidence that increases in the intracellular Ca2+ concentration may be an important regu!ator of protein kinase C activity (58-60). Glucose may, therefore, promote activation of protein kinase C in the presence of carbachol by increasing cytoplasmic Ca*+ concentrations in the vicinity of the plasma membrane. Alternatively, glucose could promote the inhibition of a protein phosphatase active toward the phosphorylated form of the 80-kDa protein kinase C substrate. Whatever the precise mechanism involved it is possible that protein kinase C plays a role in the integration of these cellular pathways in P-cells to allow the maximal insulin secretory response to the simultaneous exposure to neurotransmitter and nutrient secretagogues. In summary, the phosphorylation of the 80-kDa protein in islets has been used as a noninvasive technique for the assessment of the direct activation of protein kinase C by insulin secretagogues. The data are consistent with the involvement of protein kinase C in carbachol-induced insulin secretion and in the potentiation of glucose-induced insulin secretion but indicate that insulin secretion induced by glucose alone is mediated by mechanisms independent of protein kinase C. Acknowledgments-We wish to thank Dr. Perry Blackshear for helpful discussions at the conception of this study and for the generous gift of the MARCKS antibody. The excellent technical assistance of C. Joan Fink. Connie Marshall. and Barbara Palazuk is gratefully acknowledged. REFERENCES Prentki, M., and Matschinsky, F. M. (1987) Physiol. Reu. 67, 1185-1248 Metz, S. A. (1988) Diabetes 37, 3-7 Virji, M. A. G., Steffes, M. W., and Estensen. R. D. (1978) L?ndocrinology 102, 706-711 Malaisse, W. J.. Sener. A.. Hercheulz. A.. Carninelli. A. R.. Poloczek, P., Winand,‘J., and Castagna, M. (1980) Cancer Res:
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