Protein kinase C and insulin receptor β-subunit serine phosphorylation in cultured foetal rat hepatocytes

ELSEVIER

Molecular and Cellular Endocrinology 105 (1994) 1l-20

Protein kinase C and insulin receptor P-subunit serine phosphorylation in cultured foetal rat hepatocytes J.-L. ZachayusG*, G. Cherquib, C. Plasa ‘Laboratoire de Biologie, U.F.R. Odontologie, Universite’ Paris 7, Institut Biomidical des Cordeliers, 15, rue de I’Ecale-de-Medecine, 75270 Paris Cedex 06, France bL.uboratoire de Biologie cellulaire, INSERM-11.402, Faculte’ de Midecine Saint-Antoine, 27, rue Chaligny, 75571 Paris Cedex 12, France

Received7 March 1994; accepted 4 July 1994

Abstract

In digitonin-permeabilized cultured foetal hepatocytes, insulin receptor B-subunit was highly phosphorylated on serine residues in the presence of [Y-~*P]ATP and Ca*+, a process enhanced after short exposure to insulin with no detectable insulin receptor autophosphorylation. By contrast with this situation, experiments performed with isolated foetal insulin receptors revealed an insulin stimulation of both serine phosphorylation and tyrosine autophosphorylation. In permeabilized cells, insulin receptor /?-subunit phosphorylation was increased after a 2-rain exposure to phorbol 12-myristate 13-acetate (PMA) prior to applying the permeabilization/phosphorylation step, while it was inhibited by chronic treatment with PMA leading to protein kinase C (PKC) down modulation. The PKC specific inhibitor, GF109203X, strikingly reduced basal and insulin-enhanced phosphorylation of insulin receptor b-subunit in permeabilized cells, but failed to exert any effect with isolated receptors. Labelling of glycogen from [U14C]glucose determined 1 h after a lo-min transitory exposure to insulin and/or modulators of PKC activity showed that PMA prevented insulin glycogenic response, whereas GF109203X was ineffective. Thus, although not directly responsible for insulin receptor serine phosphorylation in cultured foetal hepatocytes, PKC physiologically regulates this process which may inhibit insulin receptor tyrosine kinase activity. This regulation is independent of the antagonistic effect of PMA-activated PKC on insulin glycogenic response. Keywords: Protein kinase C; Insulin receptor B-subunit; Serine protein phosphorylation; Glycogenesis; Permeabilized hepatocytes; Fetal rat liver

1. Introduction Phosphorylation of the insulin receptor on its #I-subunit regulates receptor tyrosine kinase activity and receptor signaIling (reviewed in Gammeltoft and van Obberghen, 1986; Htiing, 1991). Insulin induces receptor autophosphorylation on tyrosine residues, which in turn further enhances insulin receptor tyrosine kinase activity (TavarC et al., 1988; Tomqvist et al., 1988; White et al., 1988). The insulin receptor is also phosphorylatecl on serine and, to a lesser extent, on threonine residues before hormonal

stimulation in intact cells (Pang et al., 1985; White et al., 1985; Balloti et al., 1987; Issad et al., 1991; Okamoto et al., 1991). The significance of such a serine/threonine *Corresponding 21.

author. Tel. 33(l) 43 29 29 27. Fax 33 (1) 44 07 14

phosphorylation is not yet defined, no more than the identity of the kinase(s) responsible for this process. However, evidence in favour of a role for PKC comes from studies showing that activators of the kinase, such as the phorbol ester PMA, increase insulin receptor serinefthreonine phosphorylation in several cell lines such as hepatoma cells (Takayama et al., 1984) and in transfected CHO cells overexpressing both insulin receptor and PKC isoenzymes a, /?l, y (Chin et al., 1993), the latter belonging to the multigene family of related PKC isoforms (reviewed in Hug and Sure, 1993). This PMA-enhanced insulin receptor serinelthreonine phosphorylation has been correlated either with a decrease in insulin-induced insulin receptor tyrosine kinase activity (reviewed in Houslay, 1991) or with a reduced insulin receptor ability to promote activation of an endogenous substrate, the phosphatidylinositol 3-kinase complex (Chin et al., 1993).

0167-8140/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03357-Y

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After PMA treatment, insulin receptor tyrosine kinase activity assayed after insulin receptor purification has been found to be inhibited in adult hepatocytes (Caro et al., 1992) a phenomenon which is accompanied by a reduced insulin stimulation of glycogen synthase activity in FAO hepatoma cells (Takayama et al., 1984). Likewise, PKC when activated by PMA antagonizes insulin glycogenie response in ZHC cells (Caron et al., 1988). Such antagonism has been also reported in adult hepatocytes in which PMA-induced inhibition of insulin receptor tyrosine kinase activity may occur or not (Caro et al., 1992; Quentmeier et al., 1993). Cultured foetal rat hepatocytes transplanted at 18 days of gestation are highly responsive to insulin with regard to glycogenesis (Plas et al., 1979). Brief exposure to the hormone for 5-10 min, which allows the population of insulin receptors internalized after hormone binding to be recycled at the cell surface, is sufficient to trigger a clear yet not fully expressed glycogenic effect of insulin (Soubigou et al., 1986). When applying the permeabilizationlphosphorylation step in the presence of digitonin and [y-s2P]ATP for 30 mm at 4°C insulin receptor /?-subunit serine phosphorylation has been revealed as a Ca2+dependent process correlated with glycogenesis regulation (Zachayus and Plas, 1993). Thus, cultured foetal hepatocytes appear as a convenient cell system to investigate the involvement of PKC in insulin receptor serine phosphorylation and the relationship with the functioning of insulin receptor and insulin biological effect. In the present study, insulin receptor serine phosphorylation was assessed both in situ in digitonin-permeabilized cultured foetal hepatocytes where PKC had been (i) exogenously activated by short exposure to PMA, (ii) largely depleted by a chronic exposure to PMA or (iii) selectively inhibited by GF109203X (Toullec et al., 1991), and in vitro after foetal insulin receptor partial purification. The results provide evidence for the regulation of insulin receptor serine phosphorylation by PKC in situ, a process not reproduced in vitro. On the other hand, insulin-stimulated receptor tyrosine autophosphorylation which could not be detected in permeabilized foetal hepatocytes was clearly seen in partially purified foetal liver receptor preparations. PKC antagonistic effect toward insulin glycogenic response, which was observed only after exposure of cultured foetal hepatocytes to PMA, is likely not related to the action of PKC on insulin receptor phosphorylation. 2. Materials and methods 2. I. Materials Pig monocomponent insulin was from Eli Lilly. Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma. Bisindolylmaleimide (GF109203X) was purchased from Calbiochem. Phosphatidylserine, 1,Zdiolein and anti-phosphotyrosine monoclonal antibody were from

Endocrinology

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Sigma. Protein G PLUS/Protein A-agarose was from Oncogene Science. [3H]Phorbol- 12,13-dibutyrate (PDBu) (18 Ci/mmol) was from NEN. Polyclonal antibody directed against insulin receptor P-subunit C-terminal domain (last 12 amino acids) was a generous gift from Dr. C.R. Kahn. The source of other materials has been specified previously (Plas and Nunez, 1976; Zachayus and Plas, 1993). 2.2. Cell culture and treatment Primary cultures of hepatocytes were obtained from 18-day-old rat foetuses as described previously (Plas et al., 1973). After mild trypsin treatment, the isolated cells were plated on a collagen substratum to which only the hepatocytes adhered, and after 4 h the non-adhering hematopoietic cells were removed. The culture medium consisted of NCTC 109 medium (Evans et al., 1964) and supplemented with 1OpM cortisol and 10% foetal calf serum. All the experiments were performed after 2 days of culture in the presence of glucocorticoids, by which time the glycogenic effect of insulin is fully expressed (Plas and Nunez, 1976). Cell monolayers (in 3.5 cm diameter-culture dishes containing 0.6 X lo6 cells corresponding to about 210 pg protein) were incubated in the presence of the agent(s) to be tested for various times (up to 20 h) at 37°C before performing the binding and phosphoryiation studies. 2.3. Cell permeabilization and phosphorylation Enzymatic assay of phospholdephosphorylation activities in digitonin-permeabilized cells was achieved as previously described (Zachayus and Plas, 1993). Briefly, after pre-incubation of cells in conditioned medium at 37°C in the presence of the agents to be tested, culture medium was removed, cells were cooled at 4”C, washed once with cold ‘phosphorylation buffer’ (20 mM Tris, pH 7.4, 125 mM KCI, 5 mM NaCl, 5.5 mM D-ghCOSe) and incubated for 30 min at 4°C in the phosphorylation buffer containing 80pg/ml digitonin, 10 mM MgC12, 1 mM CaClz (permeabilization/phosphorylation buffer) and 30pM [Y-~~P]ATP at 120pCi/ml. Cultures were then washed once with the phosphorylation buffer supplemented with protein phosphatase inhibitors, 100 mM NaF, 10 mM NaPPi and 1 mM Na3V04, before cellular material extraction. 2.4. Analysis of 32P-labelled proteins Cells were dissolved in modified Laemmli sample buffer containing 62.5 mM Tris (pH 7.0), 2% SDS, 10% glycerol, 0.01% bromophenol blue, 50 mM dithiothreitol (DTT), 100 mM NaF, 10 mM NaPPi, 1 mM Na3V04, 2 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin and boiled for 5 min. Cell material was subjected to 10% SDS-polyacrylamide gel electrophoresis (SDSPAGE) (Laemmli, 1970) followed by autoradiography. The basis for lane loading was protein quantity, close to

J.-L. Zuchayus et al. I Molecular and Cellular Endocrinology 105 (1994) 11-20

11Opg protein per lane corresponding to the half protein material extracted from one culture. The standards used for molecular mass determination were: myosin (205 kDa), b-galactosidase (116 kDa), phosphorylase B (95 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa) and carbonic anhydrase (29 kDa). The radioactivity in 32P-labelled proteins was measured by Cerenkov counting of the corresponding gel fragment and the value obtained was subtracted by the counting of an equivalent gel fragment from the corresponding lane where no labelled band was detected.

2.5. Purification by wheat germ agglutinin chromatography of glycoproteins phosphorylated in situ After the permeabilization/phosphorylation step, cell monolayers (6 x lo6 cells) were solubilized at 4°C in 50 mM Hepes (pH 7.4), 1% Triton X-100, 10 mM NaPPi, 100 mM NaF, 1 mM Na3V0,+, 4 mM EDTA, 0.1 mg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride. Insoluble material was sedimented by centrifugation at 100 000 X g, 20 min at 4°C in a Beckman Airfuge. The lysate supernatant was applied onto a wheat germ agglutinin column (1 ml agarose packed gel). The sample was re-applied twice and then the column was washed with 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 10 mM NaPPi, 100 mM NaF, 1 mM Na3V04, 4 mM EDTA (washing solution). The glycoproteins bound to the immobilized wheat germ agglutinin were eluted with ‘washing solution’ supplemented with 300 mM N-acetyl glucosamine, and then either processed for SDS-PAGE analysis after addition of concentrated Laemmli sample buffer and boiling for 5 min, or subjected to immunoprecipitation. 2.6. [3H]PDBu binding to intact cells [3H]PDBu binding was determined according to Cherqui et al. (1990), in cells pretreated for 20 h with 0.1 or 1 pug/ml PMA or its solvent. Cells were incubated first for 20 min at 37°C in serum-free NCTC 109 medium and thereafter for 30 min at 37°C in the same medium supplemented with 20 nM [3H]PDBu. After three washings with ice-cold PBS, cells were lysed in 1 N NaOH and radioactivity was measured by liquid scintillation counting. Data were corrected for non-specific binding which was measured in the presence of lOpg/ml PMA. 2.7. Purification by wheat germ agglutinin chromatography offoetal glycoproteins to be phosphorylated in vitro

Preparations of affinity-purified insulin receptor were obtained from either 20-day-old foetal rat liver or hepatocytes transplanted from 18-day-old rat foetuses and cultured for another 2 days in the presence of glucocorticoids. Foetal rat liver membranes were first prepared according to Havrankova et al. (1978) with some modifications. 10 g of wet liver were homogenized in 50 ml of an ice-

cold solution at pH 7.6, containing

13

1 mM NaHC03,

10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 ,uM leupeptin and 0.5 mM Ca2+ (buffer A), Ca2+ being added in the present study to preserve a potential

association of PKC with the membrane fraction. After a 4-fold dilution in buffer A, the homogenate was centrifuged for 30 min at 600 X g. The supernatant was filtered through glass wool and centrifuged for 30 min at 15 000 X g. The pellet was washed once in 40 ml of

buffer A and resedimented for 30 min at 15 000 x g. Then the liver membrane pellet was resuspended in 2 ml of a buffer containing 50 mM Hepes (pH 7.4), 1% Triton X- 100, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 PM leupeptin and 0.5 mM Ca2+ (buffer B). This suspension was incubated for 2 h at 4°C and insoluble material was thereafter sedimented by centrifugation at 100 000 X g, 20 min at 4°C in a Beckman Airfuge. The supernatant was diluted threefold with a buffer containing 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM Ca2+ (buffer C), and applied onto a wheat germ agglutinin column (1 ml agarose packed gel) previously equilibrated in buffer C. The sample was re-applied twice and then the column was washed with buffer C which contained no NaCl (Smith et al., 1988). The glycoproteins bound to the immobilized wheat germ agglutinin were eluted with buffer C supplemented with 300 mM N-acetyl glucosamine. Cultured foetal hepatocyte monolayers (2.5 X 10’ cells) were directly solubilized at 4°C in buffer B. After centrifugation at 100 000 X g (20 min at 4”C), the lysate supernatant was diluted fivefold with buffer C and processed for wheat germ agglutinin chromatography as described above. In each case, the eluted fractions containing 1251-labelledinsulin-binding activity were pooled and used for phosphorylation

assays.

2.8. Phosphorylation of affinity-purified glycoproteins in vitro

Affinity-purified glycoproteins of either foetal liver (8,~g protein) or cultured foetal hepatocytes (2pg protein) were incubated in a reaction mixture containing 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM Ca2+, 10 mM MgC12, 3 mM MnC12, 1 mM DTT (buffer D), *20pglml phosphatidylserine and 8 pg/ml 1,Zdiolein with 100 nM insulin and/or 1 ,uM GF109203X or their solvent, for 40 min at 22°C to allow saturation of the receptor binding sites with the hormone. Phosphorylation was then initiated by the addition of 50pM [y-32PlATP at 6OpWml. After 20 min at 22”C, the reaction was stopped either by addition of concentrated Laemmli sample buffer and boiling for 5 min before SDS-PAGE analysis under reducing conditions, or by a 3.5-fold dilution with buffer C containing 30 mM EDTA, 5 mM EGTA, 1 mM Na3V04, 10 mM NaF and 10 mM NaPPi before immunoprecipitation.

14

J.-L. Zachayus et al. I Molecular and Cellular Endocrinology 105 (1994) 11-20

2.9. Phosphoamino acid analysis Phosphoamino acid analysis of 32P-labelled insulin receptor /?-subunit was performed by a modification (Zachayus and Plas, 1993) of the method of Cooper et al. (1983). After resolution by thin layer electrophoresis, [32P]phosphoamino acids were visualized by autoradiography, identified by comparison with native standards revealed with ninhydrin and radioactivity in phosphoserine and phosphotyrosine spots was quantitated by Cerenkov counting. 2.10. Immunoprecipitation procedure Affinity-purified glycoproteins phosphorylated in situ and in vitro were incubated overnight at 4°C with either 10pglml of anti-insulin receptor polyclonal antibody or 12 pug/ml of anti-phosphotyrosine monoclonal antibody. Protein G PLUS/Protein A-agarose was added to the immunoprecipitation reaction mixture which was incubated thereafter for 2 h at 4°C. Agarose beads were washed four times with 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 10 mM NaPPi, 10 mM NaF, 1 mM Na3V04. The immunoprecipitated proteins were solubilized in concentrated Laemmli sample buffer, boiled for 5 min and analyzed by SDSPAGE under reducing conditions. 2.11. Glycogen measurements Glycogen content and glycogen labelling from [U14C]glucose were measured as previously described (Plas et al., 1973). The following procedure has been already used to study the glycogenesis stimulation induced by a short exposure to insulin (Soubigou et al., 1986). On day 2 of culture, [U-14C]glucose (1 @i/mg) was added together with PKC modulators and/or insulin. After 10 min, the medium was replaced by an identical one except that the agents were removed. The radioactivity incorporated into glycogen was determined 1 h later. 2.12. Definitions Each protocol involved at least three independent experiments performed on different cell preparations. Data are presented as means + SD for the number of experiments indicated (n). A ‘stimulation or inhibition index’ was defined as the following ratio: nmol of glucose in glycogen/mg of cell protein in treated cultures divided by nmol of glucose in glycogen/mg of cell protein in control cultures. 3. Results 3.1. Comparison offoetal insulin receptor phosphoryhtion in situ to that in vitro In situ phosphorylation experiments were directly performed in 18-day-old cultured foetal hepatocytes grown for 2 days in the presence of glucocorticoids using a digitonin-permeabilization procedure which allows a lim-

ited number of membrane phosphoproteins to be revealed in the presence of [Y-~~P]ATP and Ca*+ (Zachayus and Plas, 1993). After purification on wheat germ agglutininagarose chromatography and SDS-PAGE analysis of the glycoproteins phosphorylated in situ, three labelled proteins of 180, 95 and 88 kDa were observed (Fig. 1A). The 95 kDa protein displayed the most important phosphorylation which was 1.4-fold stimulated after a 2-min cell exposure with insulin prior to applying the permeabilizationlphosphorylation step. This phosphoprotein was identified as the insulin receptor P-subunit by immunoprecipitation with a polyclonal antibody raised against a Cterminal peptide of the receptor P-subunit. In vitro phosphorylation experiments were performed on preparations of glycoproteins previously purified on wheat germ agglutinin from either the already considered 18-day-old cultured foetal hepatocytes or 20-day-old foetal liver. The 20-day stage of gestation was retained for foetal liver preparations since it could represent a maturation stage comparable to that of 18-day-old foetal hepatocytes after another 2 days of culture. Cell-free phosphorylation assays, when carried out with cultured foetal hepatocyte preparations, led to a 95-kDa protein labelling strikingly stimulated by insulin (Fig. 1B). Two other phosphoproteins were seen: an 85-kDa protein whose labelling was activated by insulin and a 135-kDa protein remaining phosphorylated to the same extent in the presence of the hormone. A similar protein phosphorylation pattern was observed with the foetal liver preparations, under both basal and insulin-stimulated conditions (Fig. lC), indicating that closely related kinase activities may be operative in the two preparations. With foetal liver, the 95-kDa protein whose phosphorylation was stimulated 4-fold in the presence of insulin corresponded to the insulin receptor p-subunit considering its immunoprecipitation by the anti-insulin receptor antibody. In situ phosphorylation of insulin receptor p-subunit concerned primarily serine residues under both basal and insulin-stimulated conditions, and no insulin-activated receptor autophosphorylation on tyrosine residues was detected (Fig. 2). When considering the in vitro situation, insulin receptor /?-subunit appeared to be phosphorylated under basal conditions both on serine and tyrosine residues, 59% of the 32P recovered in phosphoamino acids being present as phosphoserine and 39% as phosphotyrosine. The insulin stimulatory effect regarding insulin receptor phosphorylation in vitro was seen on both amino acid residues and particularly on the phosphotyrosine content which was enhanced 5.7-fold and then represented 87% of the 32P recovered in phosphoamino acids. 3.2. Effect of brief and chronic PMA treatments on insulin receptor,&wbunit phosphorylation in situ In digitonin-permeabilized cultured foetal hepatocytes, insulin receptor P-subunit serine phosphorylation has

J.-L. Zuchayus et al. I Molecular and Cellular Endocrinology 105 (I 994) 1 I-20

A Ins

B

in shi -

+

Anti

in Ins

+

C I

+

Ins

15

in -

+

+ Anti

IR

IR

in

10-3x

M,

Fig. 1. Comparison of foetal insulin receptor phosphorylation in situ and in vitro under basal and insulin-stimulated conditions. (A) After 2 days of culture, 18-day-old foetal hepatocytes were first incubated in conditioned medium at 37°C for 2 min in the presence of 30 nM insulin or its solvent. Then the permeabilization/labelling step was performed for 30 min at 4°C in permeabilization/phosphorylation buffer containing 30pM [y-32P]ATP (12O@i/ml), insulin being maintained during this step. Cell extracts were then processed for glycoprotein purification by wheat germ agglutininagarose chromatography. (B and C) After partial purification on wheat germ lectin as described in Section 2, insulin receptor preparations of either cultured foetal hepatocytes in B or 20-day-old foetal liver in C were pre-incubated for 40 min at 22°C in buffer D with 100 nM insulin or its solvent. Phosphorylation was then carried out for 20 min at 22°C in the presence of 50pM [r- 32P]ATP (60pCiIml). Foetal glycoproteins phosphorylatedin situ (2 pg protein, A) and in vitro (2pg protein, B and 8 pg protein, C) in the presence or absence of insulin as indicated were either directly submitted to 10% SDS-PAGE under reducing conditions or immunoprecipitated with an antibody directed against insulin receptor (Anti IR) prior to electrophoresis analysis. p, insulin receptor/I-subunit; Ins, insulin.

been revealed as a Ca2+-dependent process (Zachayus and Plas, 1993). Ca2+-activated, phospholipid-dependent con-

ventional PKC isoenzymes (reviewed in Nishizuka, 1992) are known to be exogenously activated by short exposure to PMA, a potent tumor promoter acting as a substitute In Ins

-

I

+ +

Pi

4-

P-Ser4

+-

P-Thr

+

Ins

--)

-

+PP-Tyr4

Fig. 2. Phosphoamino acid analysis of the insulin receptor &subunit phosphorylated in situ and in vitro. The 95 kDa phosphorylated protein band shown in Figs. 1A and 1C was extracted from the dried gel and phosphoamino acid determination was achieved after partial acid hydrolysis and separation using high voltage electrophoresis, followed by autoradiography. Phosphoamino acid of the insulin receptor p-subunit phosphorylated in situ and in vitro in the presence or absence of insulin (Ins) as indicated are identified from standard positions. P-ser, phosphoserine; P-thr, phosphothreonine; P-tyr, phosphotyrosine.

for diacylglycerol (Castagna et al., 1982). Foetal hepatocytes were incubated for up to 5 min at 37°C in the presence of 0.1 fig/ml PMA prior to applying the permeabilizationlphosphorylation step in Ca2+-containing buffer for 30 min at 4°C. The resulting phosphorylation state of insulin receptor p-subunit was directly visualized after whole cell extract SDS-PAGE analysis, since systematic insulin receptor purification/immunoprecipitation would require a too large supply of fetal hepatocyte cell material. The permeabilization/phosphorylation assay, under basal conditions in the absence of PMA, led to the labelling of a predominant 95kDa band (Fig. 3A) which has been described to correspond to the major phosphorylated 95kDa glycoprotein shown in Fig. 1A and identified as insulin receptor B-subunit (Zachayus and Plas, 1993). Exposure of the cells to PMA stimulated the phosphorylation of the 95kDa protein/insulin receptor p-subunit in a time-dependent way, with a maximal effect reached within 2 min (increase by 67 f 8%, n = 3), while the stimulation tended to decrease thereafter. Chronic treatment with PMA reduced to a great extent the expression of cellular PKC activity, which can be reflected by down modulation of [3H]PDBu receptor sites (Whetton et al., 1988). Phosphorylation of insulin receptor B-subunit was examined in foetal hepatocytes exposed for 20 h to 0.1 and 1 pg/ml PMA where the specific binding of r3H]PDBu to cultured cells was diminished

J.-L. Zuhuyus et al. I Moleculur und Cellular Endocrinology 105 (1994) I I-20

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B

A

a bc

e

d

f

g

11695-

*P

,‘P

66-

10'

Fig. 3. Effect of brief and chronic PMA treatments on insulin receptor /Lsubunit phosphorylation in situ. Foetal hepatocytes were first incubated either in the presence of 0.1 &ml PMA or its solvent (0.01% DMSO) for up to 5 min (A) or with increasing concentrations of PMA up to 1 pg/ml or its solvent for 20 h (B). Then the permeabilization/phosphorylation step was applied as described in the legend to Fig. 1, PMA being not maintained during this step. Cell extracts (1lOyg protein) were subjected to 10% SDS-PAGE under reducing conditions, followed by autoradiography. Control cultures (lanes a and e), cultures treated with 0.1 pg/ml PMA for 30 s (lane b), 2 in (lane c) and 5 in (lane d) or cultures treated for 20 h with 0.1 pglml (lane t) and 1 &ml PMA (lane g) are represented. #?, insulin receptor p-subunit. Three independent experiments performed on different cell preparations gave similar results.

(1410 + 80 fmol [3H]PDBu bound/mg protein for untreated cells versus 690 + 60 and 425 + 40 fmol [3H]PDBu bound/mg protein, n = 3, for cells pretreated with 0.1 and 1 pug/ml PMA, respectively). Foetal hepatocytes, made PKC-deficient by chronic PMA treatment, showed a clear dose-dependent loss of 95-kDa protein/insulin receptor p-subunit phosphorylation (inhibition by 24 f 5 and 50 & 7%, n = 3, in cells treated with 0.1 and 1 pglml PMA, respectively) (Fig. 3B). In parallel cultures, chronic PMA treatment did not alter cell-surface insulin binding when measured at 10 nM ‘2SI-labe11ed insulin (350 + 20 and 330 * 30 fmol i251-1abelled insulin bound/mg protein, n = 3, for control cells versus cells pretreated with 1 pg/ml PMA). This result rules out the possibility that the insulin receptor /?-subunit phosphorylation decrease is due to a decrease in insulin receptor binding capacity induced by long term PMA treatment. 3.3. Influence of GF109203X on foetal insulin receptor phosphorylation in situ and in vitro The bisindolylmaleimide GF109203X is a staurosporine derivative which has been described as a potent competitive inhibitor of PKC with respect to ATP and which displays a high selectivity for this kinase (Toullec et al., 1991). Considering this tool, foetal hepatocytes were incubated for 2 min at 37°C in the presence of 30 nM insulin and/or increasing concentrations of GF109203X up to

2.15 PM, the inhibitor being maintained during the permeabilization/phosphorylation step. GF109203X decreased the basal labelling of the 95-kDa protein/insulin receptor P-subunit in a dose-dependent manner, a maximal effect being obtained at a concentration close to 1 PM (inhibition by 77 + 8%, n = 3) (Fig. 4, data not shown). Insulin stimulated insulin receptor /?-subunit phosphorylation by 35 + 5%, n = 3, an enhancement which concerned serine residues as seen in Fig. 2. Insulin receptor /?-subunit phosphorylation in the presence of insulin was strikingly decreased by GF109203X so that, after exposure to the PKC inhibitor, its rate became equal to that observed in the absence of insulin. The influence of GF109203X was then assessed in vitro in order to establish whether the serine kinase activity co-purifying together with foetal liver insulin receptor and evidenced in Fig. 2 corresponded to PKC. As for other isolated receptor preparations used in the present paper, partial purification of insulin receptors on wheat germ agglutinin-agarose chromatography was achieved under conditions reported to preserve a potentially fragile association between insulin receptor and related serine kinase(s) (Smith et al., 1988). All the purification steps were performed in the presence of Ca2+ to maintain a possible association of PKC with the plasma membrane (Wolf et al., 1985). In addition, foetal liver insulin receptor phosphorylation was measured in the presence of 0.5 mM Ca2+ and of the respective co-factor and activator of PKC, phosphatidylserine (20 ,uglml) and 1,Zdiolein (8pglml). GF109203X added at a concentration of 1 ,uM did not modify either basal or insulin-enhanced insulin receptor labelling (Fig. 4). No apparent variation of the insulin receptor phosphorylation state under both basal and insulin-stimulated conditions were detected when phosphatidylserine and diolein were not added (results not shown). After immunoprecipitation by an antiphosphotyrosine monoclonal antibody, the amount of insulin receptor P-subunit phosphorylated in the presence of insulin, Ca2+ and lipids remained unchanged by the addition of GF109203X. 3.4. EfSect of short treatment with PMA and GF109203X on basal and insulin-stimulated

glycogenesis

Experiments were performed with a transitory exposure to insulin and/or modulators of PKC activity for 10 min, incorporation of [i4C]glucose into glycogen being measured 1 h later. This protocol has been described as allowing a clear yet not fully expressed glycogenic effect of insulin when present for only 10 min (Soubigou et al., 1986). PMA and GF109203X were used under conditions modifying insulin receptor p-subunit phosphorylation in permeabilized foetal hepatocytes. Addition of 10 nM insulin in the absence of these modulators produced the expected glycogenic response (insulin stimulation index: 2.0 + 0.2, n = 3) (Fig. 5). At concentrations up to 30 ng/ml PMA did not modify basal glycogen labelling,

J.-L. Zachayus et al. I Molecular and Cellular Endocrinology IO5 (1994) II-20

in Anti PY Ins

GF109203X

-

+

-

+

.

-

+

+

Ins

GF109203X

-

+

-

+

‘++I

-

-

+

+

-

+

2051169566-

10' 3x M,

Fig. 4. Influence of GF109203X on insulin receptor phosphorylation in situ and in vitro. (In situ) Foetal hepatocytes were first incubated in the pres-

ence of 30 nM insulin and/or 0.2yM GF109203X for 2 min, then tbe permeabilization/phosphorylation step was applied as described in the legend to Fig. 1, GF109203X being maintained during this step. Cell extracts (11Opg protein) were subjected to 10% SDS-PAGE under reducing conditions, followed by autoradiography. (In vitro) Purified foetaI liver insulin receptor (8,~g protein) was processed for phosphorylation assay as described in the legend to Fig. 1, except that incubations were performed in the presence of 2O@nl phosphatidylse~ne + E(pg/ml 1,24olein, 1 ,uM GF109203X being added or not. Ph~pho~la~d insulin receptor pmp~ation was either directly submi~ed to 10% SDS-PAGE under reducing conditions or immunop~cipi~~d with an ~ti-phospho~osine antibody (Anti PY) prior to efectrophoresis analysis. Phosphorylation assays performed in the presence and absence of PKC modulators and insulin (Ins) are represented. p, insulin receptor/I-subunit, while it progressively decreased the insulin glycogenic response. At concentrations higher than 30 nglrnl, PMA elicited a dose-de~ndent inhibitor effect on both basal and insulin-stimulated glycogen labellings, with an insulin glycogenic response completely abolished at 316 ng/ml PMA. The influence of GF109203X was studied with concentrations up to 1.8pM. The PKC inhibitor faiIed to exert any modification of both basal and insulinstimulated glycogenesis.

4. Discussion Insulin receptors displayed a constitutive serine phosphorylation in both permeabilized cultured foetal hepatocytes and isolated foetal insulin receptor prep~ations, although less marked in the latter situation (Zachayus and Plas, 1993; this paper). This process was stimulated by insulin in both cases. Insulin-activated receptor tyrosine autophosphorylation was not detectable in permeabilized cells but was, by contrast, strikingly pronounced after insulin receptor isolation. A comparable situation has been reported in adult liver membrane preparations where insulin stimulation of receptor autophosphorylation was not observed but could be revealed after membrane solubilization (Blackshear et al., 1984). On the other hand, a class of insulin receptors exclusively phosphorylated on serine residues in response to insulin has been

distinguished from a receptor population which bears only phosphotyrosine residues or concomitantly phosphoserine residues in Fao hepatoma cells (Pang et al., 1985) and in adult hepatocytes (Balfotti et al., 1987). Thus, the absence of insulin-activated insulin receptor tyrosine autophosphorylation in permeabilized cultured foetal hepatocytes may be linked to the high constitutive insulin receptor serine phosphorylation. An insulin-responsive serine kinase (IRSK) activity was found to be associated with the purified foetal liver insulin receptor as evidenced by its basal and insulinstimulated serine phosphorylation. An IRSK also copurifying with the insulin receptor has been reported in glycoprotein extracts from adult rat hepatocytes (Gazzano et al., 1983) and human placental membranes (Smith et al., 1988; Lewis et al., 199Ob). Previous studies concerning insulin-stimulated phosphorylation of isolated receptor from foetal liver at late stages of development have not investigated whether a foetal activity similar to that of IRSK participates to such a process (Peyron et al., 1985; Lowe et al., 1986; Sinha and Jenquin, 1987; Margolis et al., 1990). The present work provides evidence that isolated foetal insulin receptor is a substrate for a tightly associated serine kinase which is thus the foetal homologue to IRSK. Isolated foetal insulin receptor exhibited serine phosphorylation in conjunction with tyrosine autophosphory-

J.-L. Zachayus et al. I Molecular and Cellular Endocrinology 105 (I 994) I I-20

41

’

0

1

’

’

mm.I~-’ 10

’

.

c **11J 100

m c’

400

[PMA] (nglml) 41

’

I

I

0

0.1

1

2

[GF109203X](PM) Fig. 5. Effect of brief exposure to PMA and GF109203X on basal and insulin-stimulated glycogenesis. At day 2, short exposure for 10 min to 10 nM insulin and/or modulators of PKC activity in the presence of [t4C]glucose (1 @i/mg) was followed by removal of the agent(s) and further incubation in conditioned medium containing [‘4C]glucose, glycogen labelling being measured 1 h later. [14C]Glucose incorporation is represented as a function of increasing concentrations of PMA (0,O) or GF109203X (Cl,U) present for 10 min with (@,W) or without insulin (0,Cl). A representative experiment is shown, similar results being obtained with three independent experiments performed on different cell preparations. The SD values of measurements were less than 15% of the mean value.

lation under both basal and insulin-stimulated conditions. Human placenta IRSK activation requires an active insulin receptor tyrosine kinase (Smith and Sale, 1988; Baltensperger et al., 1992; Sale, 1992). On the other hand, IRSK has not been yet physically separated from insulin receptor kinase itself so that receptor B-subunit serine phosphorylation has been postulated to occur via an autophosphorylation process (Baltensperger et al., 1992). The specified IRSK of foetal liver may be also under the dependence of insulin receptor tyrosine kinase. If so, the foetal IRSK would not take part in the process of insulin receptor serine phosphorylation in cultured foetal hepatocytes which occurs in the absence of any apparent receptor tyrosine autophosphorylation. The insulin receptor constitutive phosphorylation, which has been shown to be under Ca*+-control (Zachayus and Plas, 1993), was transiently increased in situ under PKC stimulation by an acute treatment of foetal hepatocytes with PMA. This finding is in contrast with that reported in cultured adult hepatocytes where a brief exposure to PMA has no effect on constitutive insulin receptor phosphorylation (Quentmeier et al., 1993). However, it is in agreement with other studies where PMA activation of PKC promotes insulin receptor serine and

threonine phosphorylation enhancement in various cell lines (Takayama et al., 1984; Davis and Czech, 1985; Jacobs and Cuatrecasas, 1986; Hachiya et al., 1987; Koshio et al., 1989; Duronio and Jacobs, 1990; Lewis et al., 1990a; Coghlan and Siddle, 1993) including CHO transfected cells overexpressing human insulin receptor and PKC isoenzymes cz, pl and y (Chin et al., 1993). The early response of foetal hepatocytes to PMA suggests that insulin receptor /?-subunit is a substrate for PKC or for another serinelthreonine kinase at the end of a phosphorylation cascade initiated by PKC. PKC down modulation induced by chronic exposure of cultured foetal hepatocytes to PMA was correlated with a striking loss of insulin receptor P-subunit constitutive serine phosphorylation. Moreover, in the presence of the bisindolymaleimide GF109203X, which is an effective and selective inhibitor of PKC (Toullec et al., 1991; Sinnett-Smith et al., 1993) both basal and insulin-stimulated serine phosphorylation of insulin receptor were nearly abolished. In Hep G2 cells, PKC depletion or inhibition experiments suggest that PKC is not implied in constitutive and insulin-increased insulin receptor serine phosphorylation (Duronio and Jacobs, 1990). However, in CHO cells overexpressing insulin receptor, PKC may contribute to insulin-enhanced insulin receptor serine phosphorylation (Ahn et al., 1993). In foetal hepatocytes, the features of both basal and insulin-stimulated insulin receptor serine phosphorylation provided clear evidence for the involvement of PKC in these processes. Whether a particular subtype of PKC constitutively active and sensitive to insulin is responsible for insulin receptor serine phosphorylation in cultured foetal hepatocytes remains to be investigated. Several studies have suggested that PKC may be directly responsible for insulin receptor phosphorylation. Isolated human insulin receptor (Bollag et al., 1986; Lewis et al., 1990a) and its cytoplasmic domain obtained from a baculovirus expression system (Ahn et al., 1993) have been shown to serve as substrates for purified PKC. Furthermore, the formation of a tight complex between PKC and insulin receptor occurs in response to insulin in transfected cell lines (Mosthaf et al., 1993). As PKC involvement in insulin receptor serine phosphorylation in situ may originate from a close association between the kinase and insulin receptor in cultured foetal hepatocytes, insulin receptor of both cultured cells and foetal liver at an equivalent stage of gestation was purified under the selected non-stringent conditions (Wolf et al., 1985; Smith et al., 1988) in order to allow a potential copurification of PKC. Foetal IRSK activity was not modified by addition of Ca*+ + diolein + phosphatidylserine and was not affected by the PKC inhibitor, GF109203X. Similarly IRSK from human placenta does not exhibit amplified activity in the presence of the PKC co-factors and activator (Smith and Sale, 1988); it is also unable to reveal phosphorylation of

J.-L. Zachayus et al. I Molecular and Cellular Endocrinology 10.5 (1994) 11-20

a specific peptide substrate for PKC (Lewis et al., 1990b). The present data indicate that PKC does not likely participate in the IRSK activity found in foetal liver insulin receptor preparations. Despite the non-stringent conditions selected for purification, PKC failed to be copurified with foetal insulin receptor either because the association between the kinase and insulin receptor was too weak to be preserved or from the fact that PKC did not directly phosphorylate insulin receptor in cultured foetal hepatocytes. The ability for phorbol esters such as PMA to inhibit insulin activation of insulin receptor tyrosine kinase has been correlated with an increase in the b-subunit serine and threonine phosphorylation level in most cell studies (Haring et al., 1986; Takayama et al., 1988; Lewis et al., 1990a; Caro et al., 1992; Torossian et al., 1993) but not in all of them (Coghlan and Siddle, 1993; Quentmeier et al., 1993). Such inhibitory effect of PMA-activated PKC has been also suggested to be independent of insulin receptor serine phosphorylation (Anderson and Olefsky, 1991). Inhibition of insulin receptor tyrosine kinase may also be mediated by PKC translocation/activation induced by high levels of glucose (Berti et al., 1994). On the other hand, PKC plays a constitutive role in insulin receptor autophosphorylation regulation in transfected fibroblast cell lines (Pillay et al., 1990). In liver of starved rats, an increase in hepatic PKC activity is associated with stimulated insulin receptor serine phosphorylation which in turn decreases insulin receptor autophosphorylation (Karasik et al., 1990). The hypothesis of PKC involvement in insulin receptor tyrosine autophosphorylation inhibition in foetal hepatocytes is corroborated by the fact that foetal insulin receptor autophosphorylation was revealed when serine phosphorylation of the solubilized purified insulin receptor escaped PKC control. The reason why the lessening of PKC activity under GF109203X treatment did not allow the expression of insulin receptor autophosphorylation in foetal hepatocytes is not clearly understood. Whether insulin receptor-associated tyrosine phosphatases (reviewed in Goldstein, 1992; Sale, 1992) may also contribute to render insulin receptor autophosphorylation extremely evanescent remains to be elucidated. Under conditions where PKC was exogenously activated by short exposure of foetal hepatocJrt%-toPMA, insulin-stimulated glycogenesis was inhibited, basal glycogenesis being also depressed. The basal level of glycogenesis is not modified under PKC activation in ZHC cells (Caron et al., 1988) and in transfected fibroblast cell lines (Anderson and Olefsky, 1991), and is reported in adult hepatocytes to be either diminished (Car0 et al., 1992) or stimulated (Quentmeier et al., 1993). Regarding the insulin glycogenic effect, all these studies evoke that PKC after exogenous activation by PMA has the potency to antagonize the hormonal action. This may be correlated with an inhibition of glycogen synthase activity, a situa-

19

tion found in acutely PMA-treated adult hepatocytes (Roach and Goldman, 1983; Arino and Guinovart, 1986) and hepatoma cells (Takayama et al., 1984). When PKC was selectively inhibited by GF109203X, no variation in basal glycogen synthesis and insulin glycogenic response occurred in the present study. Thus, PKC is likely not involved under physiological conditions in the regulation of glycogenesis in cultured foetal hepatocytes, as postulated for ZHC cells (Caron et al., 1988). On the other hand, an endogenous stimulation of PKC activity may not be correlated with the glycogenic signal triggered by insulin. Although not directly involved in insulin receptor Bsubunit serine phosphorylation in situ, PKC physiologically regulates this process and is responsible for the inhibition of insulin receptor tyrosine autophosphorylation induction in cultured foetal hepatocytes. Also, the antagonistic effect of PKC exogenously activated by PMA on the insulin glycogenic response may be due to a direct action of the kinase on the enzymatic equipment for glycogen formation and mobilization rather than to the PKC control of insulin receptor phosphorylation which is operative in the absence of insulin. Further studies will be necessary to determine the physiological significance of PKC constitutive regulation of insulin receptor functioning in foetal hepatocytes.

Acknowledgments This investigation was supported by Communaute Economique Europeenne (contract no. SC1.223.C) and by the ‘Association pour la Recherche sur le Cancer’. We are grateful to Dr. C.R. Kahn for the generous gift of the polyclonal antibody directed against insulin receptor /3subunit. We thank Mr. E. Marie-Rose for skilful secretarial services.

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