Insulin Receptor Substrate (IRS)-2 phosphorylation is ... · Insulin promotes the dephosphorylation of glycogen synthase (GS) and consequent stimulation of glycogen synthesis [10-12].

Insulin Receptor Substrate (IRS)-2 phosphorylation is necessary for PKCζ activation by

insulin in L6hIR cells.

Francesco Oriente¶, Pietro Formisano¶, Claudia Miele¶, Francesca Fiory¶, Maria Alessandra

Maitan¶, Giovanni Vigliotta¶, Alessandra Trencia¶, Stefania Santopietro¶, Matilde Caruso¶,

Gerolama Condorelli¶, Emmanuel Van Obberghen§, and Francesco Beguinot¶*

¶Dipartimento di Biologia e Patologia Cellulare e Molecolare & Centro di Endocrinologia ed

Oncologia Sperimentale del C.N.R., Federico II University of Naples, Italy and §INSERM

Unit 145, IFR 50 Nice, France

*Address all correspondance to:

Francesco Beguinot, MD, Ph.D

Dipartimento di Biologia e Patologia Cellulare e Molecolare

Università di Napoli Federico II

Via S. Pansini, 5

80131 Naples, Italy

Phone: +39 081 7463248 Fax: +39 081 7463235

e-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 31, 2001 as Manuscript M104405200 by guest on June 29, 2020

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Running title: IRS-2 and PKCζ activation.

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ABSTRACT

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide

corresponding to the Kinase Regulatory Loop Binding Domain of IRS-2 (KRLB). In several

clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors

(IR) was accompanied by a block of IRS-2, but not IRS-1, phosphorylation and of IR binding.

GS activation by insulin was also inhibited by > 70% in these cells (P<0.001). The

impairment of GS activation was paralleled by a similarly-sized inhibition of GSK3α and

GSK3β inactivation by insulin with no change in PP1 activity. PDK1 and Akt/PKB activation

by insulin showed no difference in B2, F4 and in control L6hIR cells. At variance, insulin did

not activate PKCζ in B2 and F4 cells. In L6hIR, inhibition of PKCζ activity by either a

PKCζ antisense or a dominant negative mutant also reduced by 75% insulin inactivation of

GSK3α and β (P<0.001) and insulin stimulation of GS (P<0.002), similar to Akt/PKB

inhibition. In L6hIR, insulin induced PKCζ co-precipitation with GSK3α and β. PKCζ also

phosphorylated GSK3α and β. Alone these events did not significantly affect GSK3α and β

activities. Inhibition of PKCζ activity, however, reduced Akt/PKB phosphorylation of the key

serine sites on GSK3α and β by > 80% (P<0.001) and prevented full GSK3 inactivation by

insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle

cells. In these cells, insulin inhibition of GSK3α and β requires dual phosphorylation by both

Akt/PKB and PKCζ.

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INTRODUCTION

Most insulin effects involve tyrosine phosphorylation of insulin receptor substrates (IRSs) by

the receptor [1]. IRSs include IRS-1 and IRS-2. These proteins feature a COOH terminus

containing multiple tyrosine phosphorylation sites in various amino acid sequence motifs that

bind to the Src homology-2 domain in enzymes and adapter molecules, conveying the insulin

signal further downstream [2, 3]. In addition to the phosphorylation sites, IRS proteins contain

other domains to engage activated membrane receptors. At the NH2 terminus, the IRS

proteins contain a pleckstrin homology (PH) domain (IH1PH). The IH1PH is essential for the

physiological interaction of IRS-1 and IRS-2 with the insulin receptor [4]. In addition to the

PH domain, IRS-1 and IRS-2 contain a phosphotyrosine binding (PTB) domain (IH2PTB),

which binds the phosphorylated NPEY motif in the cytoplasmic region of the receptors for

insulin, insulin-like growth factor-1 and interleukin [3, 5, 6]. A third region, encompassing

residues 591 to 786 in IRS-2, engages the phosphorylated regulatory loop of the insulin

receptor β-subunit [7-9]. This region has been therefore termed kinase regulatory loop binding

domain (KRLB) [7-9]. Since IRS-1 does not contain a functional KRLB domain [9], we have

previously proposed that the KRLB domain might contribute to a unique signalling potential

of IRS-2 [7-9].

The signalling events by which insulin activates glycogen synthesis have become much

clearer in recent years. Insulin promotes the dephosphorylation of glycogen synthase (GS) and

consequent stimulation of glycogen synthesis [10-12]. Although glycogen synthase is a

substrate for a large number of protein kinases, the 3a-3d cluster of phosphorylation sites are

crucial to the activity of GS and these sites are phosphorylated by glycogen synthase kinase 3

(GSK3) [13, 14]. Insulin inactivates GSK3 by phosphorylation of Ser 21 (GSK3α) and/or

Ser9 (GSK3β) [13, 14] and also induces phosphorylation of the G-subunit of the glycogen-

bound form of protein phosphatase 1 (PP-1) [15]. These two events cooperate to activate GS

in the cells [16, 17] although their relative role may vary during differentiation state of

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adipocytes [17]. Insulin-dependent inactivation of GSK3 has been known to be dependent on

Akt/PKB (also known as RAC kinase) [18, 19]. Akt/PKB, in turn, was shown to be

phosphorylated in response to insulin at Thr 308 and Ser 473 [20-22], and these

phosphorylation events can be blocked by inhibitors of PI 3-kinase [10, 13, 23, 24]. PDK-1, a

phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Akt/PKB kinase, was proved to

phosphorylate Akt/PKB at Thr 308, which leads to a substantial, but incomplete activation of

Akt [21, 25]. The identity of the Ser 473 kinase is still unknown, but it is referred to as PDK-2

as it is expected that this kinase is also dependent upon PIP3. The central importance of

Akt/PKB induction for insulin inactivation of GSK3 is well established [19-21]. However,

recent evidence indicates that Akt/PKB activity is not sufficient for stimulation of GS in cells

[26]. In addition, whether induction of Akt/PKB is sufficient for insulin to inactivate GSK3 is

unknown.

In the present study, we sought to identify the molecular components of the signal

transduction pathway involved in insulin regulation of the glycogen synthetic machinery in

skeletal muscle cells, a major target of insulin action. We demonstrate that IRS-2, not IRS-1,

signals insulin activation of glycogen synthase in the L6hIR skeletal muscle cells. In these

cells, we show that insulin inhibition of GSK3 requires dual phosphorylation by both

Akt/PKB and the atypical protein kinase C PKCζ.

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MATERIALS AND METHODS

Materials - Media, sera, antibiotics for cell culture, the lipofectamine reagent, and rabbit

polyclonal antibodies toward the specific PKC isoforms were from Life Technologies, Inc.

(Grand Island, NY). Polyclonal insulin receptor antibodies were from Oncogene Science

(Mahnasset, NY), GSK3-β antibodies from Transduction Laboratories (Lexington, KY),

phosphotyrosine, IRS-1, IRS-2, Akt1/2, PDK1 and PP-1 antibodies from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA). Phospho-MAPK, phosphoser-Akt1/2, and phosphothr-

Akt1/2 were purchased from New England Biolabs (Beverly, MA). PhosphoGSK3-β

antibodies were purchased from Quality Controlled Biochemicals (Hopkintown, MA). GSK3-

α and phosphoGSK3-α antibodies, the PDK assay kit (Upstate Biotechnology cat.#17-279),

the Akt/PKB immunoprecipitation kinase assay system (Upstate Biotechnology cat. #06-558),

phospho-glycogen synthase peptide-2, recombinant GSK3-α, GSK3-β, and Akt1/PKBα were

obtained from UBI (Lake Placid, NY). The PKC assay system (cat. #V7470) was from

Promega (Madison, WI). Phosphorothioate PKCζ antisense and scrambled control

oligonucleotides have been previously reported [27, 28] and were synthetized by PRIMM

(Milan, Italy). PKCζ DN and wild-type cDNAs were generous gifts of Dr. S. Gutkind (NCI,

NIH, Bethesda). Human IRS-2 cDNA has been described in [29]. Protein electrophoresis

reagents were purchased from Bio-Rad (Richmond, VA), and Western blotting and ECL

reagents from Amersham (Arlington Heights, IL). All other chemicals were from Sigma (St.

Louis, MO).

Cell culture and transfection, and cloning of the KRLB peptide - The L6 cell clone

expressing wild-type human insulin receptors (L6hIR) have been previously characterized and

described with the term WT1 [30]. The cells were grown in DMEM supplemented with 10%

fetal calf serum, 10,000 units/ml penicillin, 10,000 µg/ml streptomycin and 2% L-glutamine,

in a humidified CO2 incubator as described by Caruso et al. [30]. Transient transfection of the

phosphorothioate oligonucleotides and of the K281->W dominant negative PKCζ mutant [31]

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was performed by the lipofectamine method according to the manufacturer's instruction. By

using pCAGGS-β-gal as a reporter, transfection efficiency was consistently between 65 and

75%, staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl b-D-

galactopyranoside.

IRS-2-(591-786) (IRS-2-KRLB ) cDNA [8, 9] was subcloned in the EcoR1 and BamH1 sites

of the pcDNA3-Myc expression vector containing the ampr selectable marker. The construct

was stably transfected in the L6hIR skeletal muscle cells using the lipofectamine method as in

[32]. Individual G418-resistant clones were selected by the limiting dilution technique (G418

effective dose 0.8 mg/ml). The expression of the KRLB peptide by the individual clones was

quantitated by Western blotting.

Western blot analysis and immunoprecipitation procedure - For these studies, cells were

solubilized in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM

Na4P2O7, 2 mM Na3VO4, 100 mM NaF, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10

µg/ml aprotinin) for 24h at 4C. Cell lysates were clarified by centrifugation at 5,000xg for 20

min, separated by SDS-PAGE and transferred on 0.45 µm Immobilon-P membranes

(Millipore, Bedford, MA) as in [7]. Upon incubation with primary and secondary antibodies,

immunoreactive bands were detected by ECL according to the manufacturer's instructions.

Immunoprecipitation of IRS-1 and IRS-2 in the cell lysates was accomplished as previously

described [7].

PKC, MAPK and PI 3-K activities - PKC activity was assayed as reported in [32]. Briefly,

for these assays, the cells were solubilized in 20 mM TRIS, pH 7.5, 0.5 mM EDTA, 0.5 mM

EGTA, 25 µg/ml aprotinin, and 25 µg/ml leupeptin (extraction buffer). Supernatants were

further centrifuged at 60,000xg for 2h and pellets solubilized with extraction buffer

supplemented with 0.5% Triton X-100. Soluble pellets were immunoprecipitated for 18h with

isoform-specific PKC antibodies and incubated with protein A-Sepharose for 2h. The

immobilized PKC was supplemented with the lipid activators (0.32 mg/ml phosphatidyl

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serine and 0.032 mg/ml diacylglycerol, final concentrations) and phosphorylation reaction was

initiated by adding the substrate solution (100mM biotinylated Neurogranin peptide, 0.5 mM

ATP, 0.25 mM EGTA, 0.4 mM CaCl2, 0.1 mg/ml BSA, 20 mM TRIS, pH 7.5, 10 mM

MgCl2, and 10 µCi/ml (3000 Ci/mmol) γ[32P]ATP, final concentrations). The reaction

mixture was further incubated for 30 min at room temperature, and phosphorylation reaction

was terminated by adding 7.5M guanidine hydrochloride and spotting on phosphocellulose

discs. Disc-bound radioactivity was quantitated by liquid scintillation counting.

Determinations of PKCδ and PKCζ activities using either the AcMBP(4-14) peptide as

substrate, or the H-Arg-Phe-Ala-Val-Arg-Asp-Met-Arg-Gln-Thr-Val-Ala-Val-Gly-Val-Ile-

Lys-Ala-Val-Asp-Lys-Lys-OH peptide (for PKCδ) or the PKCε pseudosubstrate region (for

PKCζ,) provided consistent results.

MAPK was assayed as previously described [32]. Briefly, cell lysates (200 µg protein/assay)

were immunoprecipitated with MAPK antibodies and then incubated with protein A-

sepharose for 2h. Immobilized MAPK was washed three times with ice-cold TAT buffer (50

mM HEPES, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, 10%

glycerol, 1% Triton X-100), twice more with HNTGVa buffer (50 mM HEPES, pH 7.5, 150

mM NaCl, 2 mM Na3VO4, 10% glycerol, 1% Triton X-100) and then resuspended in

HNTGVa supplemented with 60 mM Mg acetate, 30 µM ATP, 6 mM DTT, 1 µg/ml myelin

basic protein (MBP), and 0.5 µCi [γ32P]ATP. Upon incubation for 30 min at 25C, reaction

mixtures were spotted on phosphocellulose discs, washed three times with 1% (v/v)

phosphoric acid and once more with ethanol. Disc-bound radioactivity was quantitated by

liquid scintillation counting. For quantitation of PI 3-kinase activity, the cells were solubilized

in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10% EDTA, 10

mM Na4P2O7, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, 1 mM

phenylmethylsulfonyl fluoride (TAN buffer). Aliquots of the lysates were precipitated with

IRS-1, IRS-2 or phosphotyrosine antibodies and PI 3-kinase activity determined as reported in

[7].

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PDK1, Akt/PKB, PP1, GSK3 activity and GSK3 phosphorylation - For these studies, the

cells were solubilized in 50 mM TRIS, pH 7.5, 0.1% Triton X-100, 1 mM EDTA, 1 mM

EGTA, 50 mM NaF, 10 mM sodium glycerophosphate, 5 mM Na4P2O7, 1 mM Na3VO4,

0.1% 2-mercaptoethanol, 1 µM microcystin LR, 0.2 mM PMSF, 25 mg/ml aprotinin

(extraction buffer). Cell lysates were clarified by centrifugation at 5,000 x g for 20 min and

immunoprecipitated with either PDK1 or Akt1/PKB antibodies. PDK1 and Akt/PKB activities

were then measured in the immunoprecipitates using the Upstate Biotechnology kinase assay

kits and according to the manufacturer instructions.

For determination of PP1 activity, the cells were scraped in PP1 homogenization buffer (50

mM HEPES, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, 10%

glycerol, 0.5% Triton X-100) supplemented with 0.2 mM phenylmethylsulfonyl fluoride and

25 mg/ml aprotinin. The lysate was sonicated on ice and precipitated with PP1 antibodies.

Immunocomplexes were immobilized on protein A sepharose, washed twice with 50 mM

HEPES, pH 7.4 and diluted in 25 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM EDTA and 4.5

nM okadaic acid. Samples were then incubated at 37C for 15 min and the reaction was

initiated by addition of 15 µg of [32P]-labelled phosphorylase α in the presence of 3 nM

okadaic acid and 5 mM caffeine. Phosphate release was determined as reported by Lazar et al.

[33].

For quantitating GSK3 activity, cells were solubilized in 50 mM TRIS, pH 7.5, 1 mM EDTA,

1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X-100, 5 mM Na4P2O7,

10 mM sodium glycerophosphate, 0.2 mM PMSF, 25 µg/ml aprotinin, 25 µg/ml leupeptin,

clarified by centrifugation at 5,000 x g for 20 min and immunoprecipitated with either GSK3-

α or GSK3-β antibodies. Immunocomplexes were immobilized on protein G-Sepharose,

washed twice with 50 mM TRIS, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4,

0.1% 2-mercaptoethanol, 1% Triton X-100, 5 mM Na4P2O7, and 10 mM sodium

glycerophosphate, and once more with reaction buffer (8 mM MOPS, 0.2 mM EDTA and 10

mM Mg acetate). Pellets were resuspended in 20 µl of reaction buffer containing 250 mM

Phospho-Glycogen Synthase Peptide-2, 500 mM ATP, 75 mM MgCl2, and 1µCi/µl (3,000

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Ci/mmol) [γ-32P]ATP and further incubated at 30C. Reaction mixtures were spotted on

phosphocellulose discs, washed three times with 0.75% phosphoric acid and once more with

acetone. Disc-bound radioactivity was quantitated by liquid scintillation counting. In some of

the experiments, purified (0.1µg) rather than immunoprecipitated GSK3α or GSK3β were

used. In vitro phosphorylation of GSK was analyzed by incubating GSK3α or GSK3β

immunoprecipitates with 0.1 mg recombinant PKCζ in the presence of 0.5 mM ATP, 0.25

mM EGTA, 0.4 mM CaCl2, 0.1 mg/ml BSA, 20 mM TRIS, pH 7.5, 10 mM MgCl2, 0.32

mg/ml phosphatidylserine and 10 µCi/ml (3,000 Ci/mmol) [γ-32P]ATP (final concentrations).

The incubation was prolonged for 30 min at room temperature and then stopped by addition of

Laemli sample buffer. Phosphorylated proteins were separated by SDS-PAGE and identified

by autoradiography. In some of the experiments, purified (0.1 µg), rather than

immunoprecipitated, GSK3α and GSK3β were used in the phosphorylation assays.

Thymidine incorporation and glycogen synthase assays - The thymidine incorporation

assay was accomplished as previously reported [34]. Briefly, L6hIR myoblasts were seeded in

six-well plates and, after 18h, feeded with DMEM supplemented with 0.25% BSA. The cells

were further incubated for 16h in the absence or the presence of 100 nM insulin, followed by

addition of 500 nCi/ml of [3H]thymidine. 4h later, the cells were washed with ice-cold 0.9%

NaCl and then with ice-cold 20% TCA followed by solubilization with 1N NaOH.

Radioactivity was quantitated by liquid scintillation counting. Glycogen synthase activity was

assayed as described in [34].

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RESULTS

IRS-2 block by expression of the KRLB peptide in L6hIR cells - The cDNA fragment

corresponding to aminoacids 591-786 of IRS-2 was transfected in L6hIR skeletal muscle

cells. This fragment encodes the complete Kinase Regulatory Loop Binding (KRLB) domain

of IRS-2 protein [7-9]. Several clones of L6hIR cells stably expressing the KRLB peptide

were selected and characterized and two of these clones, expressing lower (B2) and higher

levels of the peptide (F4), were studied in detail (Fig.1A). In extracts from insulin-exposed

L6hIR cells, the expressed KRLB peptide co-precipitated with insulin receptors (Fig.1B). The

co-precipitation was dependent on the concentration of insulin to which the cells were

exposed (insulin ECmax 100 nM, EC50 5 nM), as well as on the KRLB peptide expression

levels in the cells. In extracts from B2 and F4 cells, the KRLB peptide-insulin receptor

interaction was accompanied by 75 and 95% decreased IRS-2 co-precipitation with the

insulin receptor, respectively, as compared with those occurring in cells transfected with the

plasmid alone (Fig.1C). Expression of the KRLB peptide at increasing levels also caused

progressive decrease in insulin-dependent phosphorylation of IRS-2, with no change in the

IRS-2 content of the cells (Fig.2A). Decreased IRS-2 phosphorylation by the KRLB peptide

corresponded to parallel increases in receptor co-precipitation of IRS-1 (Fig.1C) as well as in

insulin-dependent tyrosine phosphorylation of IRS-1 (Fig.2B). As was the case for IRS-2,

IRS-1 levels were unchanged in the KRLB expressors as compared to L6hIR cells transfected

with the empty vector.

Insulin action in cells expressing the KRLB peptide - To address the functional

consequences of blocking IRS-2-mediated insulin signalling in L6hIR cells, we have

compared proliferative and glycogen synthetic responses in KRLB expressors and

untransfected cells. As shown in Fig.3 (top panel), basal (non insulin-dependent)

incorporation of thymidine was increased by 25 and 40% in B2 and F4 cells, respectively,

compared to control cells transfected with the vector alone or the untransfected cells (P<

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0.001). Maximal insulin-stimulated thymidine incorporation was also increased by 18 and

40% in the B2 and F4 cell clones, respectively (P< 0.001). Increased thymidine incorporation

into DNA in these cells was paralleled by 18 and 30% increased basal (P< 0.05) and a

similarly sized (P< 0.05) increase in insulin-stimulated MAPK activity compared to the

control cells (Fig.3, bottom panel; P< 0.001). At variance with proliferative responses, basal

glycogen synthase activity was unchanged in B2 and F4 cells (Fig.4A). In the L6hIR control

cells, insulin increased glycogen synthase activity in a dose and time-dependent fashion. Half-

maximal and maximal insulin effects were achieved at 5 and 100 nM, respectively (Fig.4A).

Also, maximal stimulation was achieved within 30 min after insulin addition (Fig.4B).

Insulin-stimulated glycogen synthase activity was inhibited by 70 and 85% in B2 and F4 cells,

respectively. Immunoprecipitated GSK3α and GSK3β activities toward the specific substrate

Phospho-Glycogen Synthase Peptide 2 were also not different in basal F4 and in control cells

(Fig.5A). In the F4 cells, insulin, either at 1nM or at 100 µM, did not induce any significant

inhibition of either GSK3α or GSK3β (Fig.5A). At these same concentrations, insulin elicited

20 and 40% inhibition of GSK3α activity and 25 and 60% inhibition of GSK3β in the control

cells (transfected with the empty vector). Phosphorylation of GSK3α and β also exhibited no

change after insulin stimulation of the F4 cells, while increasing in a dose-dependent manner

in the L6hIR cells (Fig.5A, inset). Similar results were obtained with the B2 cell clone (data

not shown). Phosphatase activity in PP-1 immunoprecipitates from L6hIR cells was well

detectable, but increased by only 25% (P< 0.05) upon insulin exposure of the cells (Fig.5B).

No significant difference in PP-1 protein expression and activity were observed in the F4

compared to the control cells, whether in the absence or the presence of insulin.

PKCζ activation in cells expressing the KRLB peptide - To elucidate the insulin signalling

events responsible for the block of glycogen-synthetic responses in the KRLB peptide

expressing cells, we have first analyzed IRS-1 and IRS-2-associated PI 3-K activity. Basal

IRS-1-coprecipitated PI 3-kinase in B2 and F4 cells featured 20 and 40% increased levels

compared to that in control cells (Fig.6, top panel; P <0.05). Maximal insulin-stimulated PI 3-

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kinase activity associated to IRS-1 also showed 30 and 45% increase in cells expressing the

KRLB peptide (P< 0.05), paralleling IRS-1 tyrosine phosphorylation. The PI 3-kinase activity

measured in IRS-2 immunoprecipitates from basal B2 and F4 cells showed 40 and 60% lower

levels as compared to control cells (Fig.6, middle panel; P< 0.001). Insulin-stimulated activity

of PI 3-kinase associated to IRS-2 was inhibited by 80 and 95% in B2 and F4 cells,

respectively. Total PI 3-kinase activity associated with tyrosine phosphorylated proteins

featured no significant differences in the B2, the F4 and the control cells, however (Fig.6,

bottom panel). It appeared therefore that IRS-1 and IRS-2 are redundant in transducing insulin

activation of PI 3-kinase in L6hIR as in other models [32, 35].

Known proteins mediating insulin signals downstream PI 3-kinase include PDK1, Akt/PKB

and PKCζ [13, 14, 36]. Insulin-dependent activation of PDK1 and Akt/PKB were unchanged

in the F4 compared to control cells (Fig.7A). Insulin-dependent phosphorylation of key

phosphorylation sites on Akt/PKB (Ser473 and Thr308) were also unchanged in the F4

compared to control cells (Fig.7B). Similarly, protein levels of both PDK1 and Akt/PKB were

unaffected by expression of the KRLB peptide. In L6hIR control cells, insulin induced PKCα,

β, δ and ζ in a dose-dependent fashion. In parallel with IRS-2 phosphorylation, however,

maximal insulin-stimulated PKCζ activation (2-fold vs. basal) was inhibited by 70, and >95%

in the B2 and the F4 cells, respectively (Fig.8C, bottom panel). PKCζ inhibition occurred

with no detectable changes in the expression of PKCζ protein (Fig.8A) or mRNA (data not

shown) in the cells. The expression of PKCδ was also unchanged in the KRLB peptide

expressing cells, although maximal insulin-dependent activation was reduced by 20% (P<

0.05; Fig.8C, top panel). At variance, PKCα and PKCβ expression and insulin-dependent

activities were not significantly different in the KRLB and the control cells (Fig.8A,B).

The specific role of IRS-2 in insulin activation of PKCζ and glycogen synthetic responses was

further addressed by transient transfection of IRS-2 cDNA in L6hIR cells. As shown in

Fig.9A, overexpression of IRS-2 (10-fold vs. control cells) was accompanied by a constitutive

increase in PKCζ activity levels. Insulin further increased PKCζ activity by 40% in cells

overexpressing IRS-2. The changes in PKCζ activity levels caused by IRS-2 overexpression

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were closely paralleled by very similar changes in glycogen synthase activity (Fig.9B) and

GSK3α and β phosphorylation (Fig.9C).Thus, the block of insulin activation of PKCζ and of

glycogen synthetic responses in cells expressing the KRLB peptide seemed to be caused by a

specific block of IRS-2 signalling.

Effects of PKCζ block in L6hIR muscle cells - We tested the hypothesis that PKCζ is also

involved in insulin activation of glycogen synthase in muscle cells. To address this issue, we

transfected a PKCζ antisense oligonucleotide (PKCζ-AS) in wild-type L6hIR skeletal muscle

cells. This antisense reduced PKCζ protein levels by >70% in the cells, without affecting

those of PKCδ (Fig.10A). Activation of PKCζ by insulin was also inhibited by about 70% by

the PKCζ antisense (Fig.10C). In parallel, PKCζ-AS transfection blocked insulin inhibition of

GSK3α and β by >75% (P<0.001; Fig.10B). Control oligonucleotides (PKCζ-S) neither

decreased PKCζ (or PKCδ) levels nor GSK inhibition by insulin as compared to cells not

treated with the antisense. In addition, we transfected the dominant negative K281->W PKCζ

mutant (PKCζ-DN) in L6hIR cells. Expression of this mutant, at 50-fold higher levels than

endogenous PKCζ, significantly reduced insulin activation of PKCζ (P< 0.001; Fig.10C). As

in cells transfected with the PKCζ antisense, the lack of PKCζ insulin response in PKCζ-DN

cells was accompanied by inhibition of insulin effect on GSK3α and β (P< 0.001). Similar to

GSK3, insulin activation of glycogen synthase was largely prevented by PKCζ-AS (though

not PKCζ-S) treatment and by transfection of the PKCζ-DN (Fig.11). Different from PKCζ,

however, inhibition of PKCδ activity with Rottlerin (3µM) had no effect on either glycogen

synthase activation by insulin or GSK3α and β inhibition (data not shown). This indicated that

insulin effects on GSK3 and glycogen synthase in L6hIR skeletal muscle cells requires PKCζ

not PKCδ.

PKCζ association and phosphorylation of GSK3α and GSK3β - To further investigate the

role of PKCζ in activating the glycogen synthetic machinery in L6hIR cells, we sought to

identify potential PKCζ-GSK3 interactions. Insulin pre-treatment of intact L6hIR cells was

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found to induce co-precipitation of PKCζ with GSK3α and GSK3β in the cell lysates

(Fig.12A). PKCζ co-precipitation with GSK3α and β was detectable by either blotting PKC

precipitates with GSK3 antibodies or vice-versa (data not shown). The dominant negative

PKCζ-DN mutant did not co-precipitate with either GSK3α or GSK3β, either in the absence

or the presence of insulin, indicating PKCζ activation is necessary for its association with

GSK3. In vitro, activated recombinant PKCζ phosphorylated purified GSK3α and GSK3β

(Fig.12B,C). Phosphorylation also occurred using GSK3α and GSK3β preparations from

L6hIR cells (data not shown). PKCζ phosphorylation of GSK3α and GSK3β, however, did

not reduce GSK3 activities toward the Phospho-Glycogen Synthase Peptide 2 substrate

(Fig.13A). This suggested that, while necessary, PKCζ activation may not be sufficient to

inhibit GSK3α or β upon exposure of intact cells to insulin.

PKCζ inhibition of GSK3α and GSK3β - In most cell types, phosphorylation by Akt/PKB is

believed to be necessary for insulin to restrain GSK3α and GSK3β activities [18]. Consistent

with those previous findings, treatment of L6hIR and F4 cells with the Akt/PKB inhibitor

ML-9 reduced insulin effect on GSK3α and β as well as on glycogen synthase activity by

>80% (P< 0.001; Fig.14).Thus, we tested the hypothesis that phosphorylation of GSK3 by

both PKCζ and Akt/PKB is required for fully inhibiting GSK3 activity in L6hIR cells. To

address this possibility, we first phosphorylated in vitro purified GSK3α and β with PKCζ.

We then incubated PKCζ-phosphorylated GSK3 preparations with immobilized Akt/PKB

from either basal or insulin-treated cells, or with an active recombinant Akt/PKB. As shown

in Fig.13A, incubation of PKCζ-phosphorylated GSK3α with the activated Akt/PKB

preparations reduced GSK3α activity by > 70%. At variance, Akt/PKB phosphorylation,

alone, elicited only a 35-40% inhibition of GSK3α, in the absence of PKCζ pre-treatment. No

significant inhibition of GSK3α activity occurred upon incubation with inactive Akt/PKB,

both in the absence or in the presence of previous PKCζ phosphorylation. Same results were

obtained with GSK3β (data not shown). At variance with Akt/PKB, PKCζ did not

phosphorylate Ser21 on GSK3α and Ser9 on GSK3β in vitro (Fig.13B). However, a 2.5-fold

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increase in Akt/PKB-induced phosphorylation of Ser21 and Ser9 was detectable when GSK3α

and β were incubated with both PKCζ and Akt/PKB.

To address the relevance of PKCζ phosphorylation to GSK3 control by Akt/PKB in vivo, we

analyzed the key Akt/PKB phosphorylation sites on GSK3α and β in cells transfected with

either PKCζ antisense, or the dominant negative PKCζ-DN mutant. In both PKCζ antisense

transfected cells and in cells overexpressing the PKCζ dominant negative mutant, Ser21

phospho-GSK3α and Ser9 phospho-GSK3β were almost absent compared to the untransfected

cells, either in the absence or in the presence of insulin (Fig.13C,). This paralleled the lack of

insulin effect on GSK3 activity occurring in cells when PKCζ expression or function is

blocked. Thus, PKCζ phosphorylation appears to be necessary and permissive for further

phosphorylation of GSK3α and β by Akt/PKB as well as for insulin constrain of GSK3α and

β activities.

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DISCUSSION

In the present report, we describe a novel approach to investigate IRS-2- mediated events in

insulin activation of glycogen synthase in cultured cells. We have expressed a peptide

corresponding to the Kinase Regulatory Loop Binding (KRLB) domain of IRS-2 (aminoacids

591-786 [7-9]) in L6hIR skeletal muscle cells. Consistent with our previous in vitro data [7-

9], expression of this peptide specifically blocked IRS-2 association and phosphorylation by

the active insulin receptor kinase. It appears, therefore, that the KRLB domain is necessary for

enabling IRS-2 to interact with the insulin receptor in intact cells as well as in vitro.

Block of IRS-2 binding to the receptor by the KRLB peptide was accompanied by increased

receptor binding and phosphorylation of IRS-1, with no change in IRS-1 protein levels. The

KRLB domain is unique to IRS-2. In addition to the KRLB however, IRS-2 possesses

Pleckstrin homology (PH) and Phosphotyrosine binding (PTB) domains homologous to those

enabling IRS-1 to interact with the insulin receptor [3]. Prevention of IRS-2 binding to the

receptor through the expression of the KRLB peptide may remove IRS-2 competition for PH

and PTB binding sites on the receptor, fostering binding of IRS-1. Consistent with this

possibility, muscles from IRS-1 knockout mice and L6hIR cells transfected with IRS-1

ribozyme have also been reported to feature increased phosphorylation of IRS-2 [32, 35].

Expression of the KRLB peptide was accompanied by increased insulin mitogenic activity

through the MAPK system but block of insulin activation of the glycogen synthetic apparatus.

In addition, IRS-2 overexpression constitutively activated GSK3 phosphorylation and

glycogen synthase activity in L6hIR cells. Thus, IRS-1 seems to transduce insulin mitogenic

effects, while IRS-2 is the main molecule involved in glycogen synthetic responses in L6hIR

muscle cells. In these same cells, ribozyme block of IRS-1 expression prevents insulin

mitogenic but not glycogen synthetic responses [32]. Also, the expression of an insulin

receptor mutant (IR1152), featuring constitutively increased phosphorylation of IRS-2,

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induces constitutive activation of the glycogen synthetic apparatus in the L6 cells [7]. This

effect can be prevented by expressing the KRLB peptide in the cells (data not shown). In

addition to the muscle cells, IRS-2 signalling appears to be prominent in transducing insulin

activation of the glycogen synthetic apparatus in isolated liver cells [37]. Thus, the prominent

role of IRS-2 in insulin activation of glycogen synthesis may be common to the two major

tissues accomplishing this function in the organism.

Akt/PKB activation is known to represent a major mechanism leading insulin to stimulate

glycogen synthase activity in cells [13, 21]. Accordingly, treatment of L6hIR cells with the

Akt/PKB inhibitor ML-9 [38] prevents insulin stimulation of glycogen synthesis. In the

present paper, however, we report that Akt/PKB phosphorylation and activation occurs at

normal levels in cells expressing the KRLB peptide, despite the block of insulin-stimulated

glycogen synthase activity. This indicates that Akt/PKB activation is necessary but not

sufficient for enabling insulin control of glycogen synthase in L6hIR cells. PDK1 and PDK2

phosphorylation of Akt/PKB in response to insulin also occurred at the same levels in cells

expressing the KRLB peptide and in untransfected cells. Thus, in cells expressing the KRLB

peptide, increased IRS-1 phosphorylation is paralleled by a similar increase in MAPK activity

and insulin mitogenic signalling but unchanged PDK1 and Akt/PKB activities. This may

occur because of the following possibilities: i., insulin activation of PDK1 and Akt/PKB (at

variance with that of MAPK) is already saturated by IRS-1 signalling in untransfected cells;

ii., IRS-1 and IRS-2 may be redundant in signalling activation of PDK1 and Akt/PKB, but not

of MAPK activation, so that increased IRS-1 phosphorylation in KRLB expressing cells

exactly compensates for lack of IRS-2 phosphorylation in activating PDK1 and Akt/PKB but

not MAPK; iii., insulin activation of PDK1 and Akt/PKB, different from that of MAPK, may

not require IRS-1-/ or IRS-2-associated PI 3-kinase, as recently proposed by Whitehead et al.

[39]. These possibilities are currently under investigation in our laboratory.

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At variance with PDK1 and Akt/PKB, insulin induction of PKCζ activity was blocked in cells

expressing the KRLB peptide, indicating a major role of IRS-2 in signalling PKCζ activation.

It is possible that expression of the KRLB peptide reduces the amount of phosphatidylinositol

3-phosphate produced after insulin stimulation of the cells and that PDK1, Akt/PKB and

PKCζ feature a differential need for phosphatidylinositol 3-phosphate for activation. This

hypothesis is unlikely to explain the differential activation of PDK1, Akt/PKB and PKCζ in

KRLB and control cells, since the total amount of PI 3-kinase activity associated with tyrosine

phosphoproteins was not different in the two cell types. Alternatively, activation of PKCζ by

IRS-2 associated PI 3-kinase may occur in an intracellular compartment different from that

where PDK1 and Akt/PKB are activated by IRS-1 associated PI-3 kinase. Consistent with this

possibility, tyrosine phosphorylated IRS-1 and IRS-2 are differentially located inside the cell

[40, 41]. Other PI 3-kinase docking substrates, such as IRS-4 and GAB, may also be

differentially phosphorylated by insulin in cells expressing the KRLB peptide, and contribute

to the different activation of PDK1, Akt/PKB and PKCζ. This latter possibility is presently

under investigation in our laboratory.

We have also shown that antisense inhibition of PKCζ expression or block of PKCζ activity

with a dominant negative PKCζ mutant prevents insulin effect on GSK3 (both α and β) and

on glycogen synthase, as in the case of Akt/PKB block. It appears therefore that both PKCζ

and Akt/PKB are necessary for insulin control of GSK3 and glycogen synthase in intact

L6hIR cells. In these same cells, antisense block of PKCζ expression or inhibition of PKCζ

activity with a dominant negative PKCζ mutant abolished insulin phosphorylation of the key

Akt/PKB phosphorylation sites in GSK3α and β (Ser21 and Ser9, respectively). These sites

might undergo promiscuous phosphorylation by PKCζ in the L6hIR cells. This is an unlikely

possibility, however, since, in vitro, PKCζ does not phosphorylate either GSK3α on Ser21 or

GSK3β on Ser9. Alternatively, PKCζ phosphorylation of GSK3α and β may be permissive for

phosphorylation and inactivation by Akt/PKB. In vitro, recombinant PKCζ phosphorylates

GSK3α and β but is unable to inhibit its activity. Recombinant Akt/PKB, alone, exhibits only

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modest effects on GSK3 activity. We have found, however, that PKCζ phosphorylation of

GSK3α or β enables Akt/PKB to further inhibit the GSK3s in vitro. This sequential

phosphorylation of GSK3 by PKCζ and Akt/PKB was accompanied by almost complete block

of GSK3 activity. Thus, in vitro, full inhibition of GSK3α or β activities requires

phosphorylation by both PKCζ and Akt/PKB. We propose therefore that dual phosphorylation

of GSK3α and β by PKCζ and Akt/PKB may also be necessary for full inactivation of GSK3

by insulin, at least in intact L6hIR muscle cells. It appears that PKCζ is involved in

transducing insulin action to GSK3 and glycogen synthase in addition to regulating insulin-

mediated glucose uptake and general protein synthesis [42-44].

Phosphorylation of GSK3 by Akt/PKB is believed to represent a major mechanism

responsible for insulin control of GSK3 and glycogen synthase activities in cells. This is also

the case for the L6hIR myotubes, since insulin elicited only a slight effect on PP-1 in these

cells. To our knowledge, however, the present report provides the first evidence that PKCζ

phosphorylation of GSK3 is permissive for insulin-dependent Akt/PKB regulation of GSK3.

Mapping the relevant PKCζ phosphorylation sites on GSK3 is presently in progress in our

laboratory.

A recent report by Tsujio et al. showed that activation of PKCδ rather than ζ is involved in

insulin signalling to GSK3 in neuroblastoma cells [45], suggesting that different PKC

isoforms may accomplish this function in different cell types. In addition, in liver, PKCζ is

not involved in insulin signalling to glycogen synthase [10]. Also, the expression of the KRLB

peptide in mouse liver cells inhibits insulin activation of Akt/PKB despite the unchanged

induction occurring in the L6hIR muscle cells (our unpublished observation). Thus, the

molecular mechanisms responsible for insulin inactivation of GSK3 may feature tissue

specificity.

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ACKNOWLEDGEMENTS

The Authors are grateful to Dr. E. Consiglio for his continuous support and advice during the

course of this work. We also like to thank Dr. L. Beguinot (DIBIT, H.S. Raffaele, Milan) for

advice and critical reading of the manuscript, and Dr. D. Liguoro for the technical help.

This work was supported in part by the European Community (Grant QLRT-1999-00674),

grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to F.B. and P.F., the

Ministero dell' Università e della Ricerca Scientifica (F.B.), and the C.N.R. Target Project on

Biotechnology (F.B.). The financial support of Telethon - Italy (Grant n. 0896 to F.B.) is

gratefully aknowledged.

G. Vigliotta and M.A. Maitan are recipients of fellowships of the Federazione Italiana per la

Ricerca sul Cancro (FIRC).

Drs. P. Formisano and F. Oriente contributed equally to the present manuscript.

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FIGURE LEGENDS

Fig.1 KRLB peptide expression in L6hIR cells. A - L6hIR cells were stably transfected with

the myc-tagged KRLB cDNA, as described under Materials and Methods. Clones of cells

expressing the KRLB peptide (A4,B2,F4,F5) were then lysed, separated by SDS-PAGE,

blotted with myc antibodies, and compared with control cells (L6hIR). F4 and B2 cells were

stimulated with 100 nM insulin for 5 min. as indicated and then immunoprecipitated with

insulin receptor antibodies, followed by blotting with either myc antibodies (B) or IRS-1 or

IRS-2 antibodies (C). Bands were revealed by ECL as specified under Materials and Methods.

The autoradiographs shown are representative of three (A,B) and four independent

experiments.

Fig.2 Effect of KRLB peptide expression on IRS-2 and IRS-1 phosphorylations. L6hIR

(control), B2, and F4 cells were treated with 100 nM insulin as indicated. The cells were

precipitated with IRS-2 (A) or IRS-1 antibodies (B) followed by blotting with

phosphotyrosine antibodies (PTYR) ECL and autoradiography. The same filters were

subsequently re-probed with IRS-2 (A) or IRS-1 (B) antibodies and subjected to ECL and

autoradiography. The autoradiographs shown are representative of three independent

experiments.

Fig.3 Insulin mitogenic effects in cells expressing the KRLB peptide. L6hIR, B2 and F4

cells were incubated with 100 nM insulin for 16h (thymidine incorporation) or 5 min (MAPK

activity) as indicated. Thymidine incorporation into DNA and MAPK activity toward MBP

were then determined as described under Materials and Methods. Bars represent the mean ±

S.D. of triplicate determinations in four (thymidine incorporation) and five (MAPK activity)

independent experiments.

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Fig.4 Glycogen synthase activity in cells expressing the KRLB peptide. A - L6hIR, B2 and

F4 cells were exposed to the indicated concentrations of insulin for 30 min. Glycogen

synthase activity was then assayed as reported under Materials and Methods. Each data point

is the mean ± S.D. of duplicate determinations in four independent experiments. B - The cells

were exposed to 100 nM insulin for the indicated times and glycogen synthase activity

assayed as above. Each data point is the mean ± S.D. of duplicate determinations in three

independent experiments.

Fig.5 GSK3α and β and PP1 activities in cells expressing the KRLB peptide. A - L6hIR and

F4 cells were exposed to the indicated concentrations of insulin, lysed and precipitated with

GSK3α or β antibodies. Kinase activity toward the Phospho-glycogen Synthase Peptide-2 was

then assayed in the immunoprecipitates as described under Materials and Methods. For

control, aliquots of the lysates were also blotted with Ser21phospho-GSK3α or Ser9phospho-

GSK3β antibodies (insets). B - PP1 activity was also assayed in immunoprecipitates from

lysed cells as described under Materials and Methods. For control, aliquots of the lysates were

blotted with PP1 antibodies. Bars represent the mean ± S.D. of duplicate determinations in

four (A) and five (B) independent experiments.

Fig.6 IRS-1 and IRS-2 associated PI 3-kinase activities in cells expressing the KRLB

peptide. L6hIR, B2 and F4 cells were stimulated with the indicated concentrations of insulin,

lysed and precipitated with either IRS-1 (top panel), IRS-2 (middle panel) or phosphotyrosine

antibodies (bottom panel). PI 3-K activity was assayed in the immunoprecipitates as described

in [25]. Bars represent the mean ± S.D. of duplicate determinations in three (top and bottom)

and four (middle) independent experiments.

Fig.7 Insulin activation of PDK1 and Akt/PKB in cells expressing the KRLB peptide. A -

L6hIR and F4 cells were stimulated for 5 min with 100 nM insulin as indicated, solubilized,

and immunoprecipitated with PDK1 or Akt/PKB antibodies. PDK1 and Akt1/2 activities were

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then assayed in the immunoprecipitates as described under Materials and Methods. Bars

represent the mean ± S.D. of duplicate determinations in four (PDK1) and three (Akt/PKB)

independent experiments. B - Aliquots from the lysates were blotted with PDK1 or Akt/PKB

antibodies revealed by ECL and autoradiographed. Upon autoradiography, Akt/PKB filters

were re-probed with phosphoSer423-Akt/PKB, or phosphoThr308-Akt/PKB antibodies and

revealed as above. The autoradiographs shown are representative of four (PDK1) and three

(Akt/PKB) independent experiments.

Fig.8 PKC activation by insulin in cells expressing the KRLB peptide. B,C - L6hIR, B2 and

F4 cells were stimulated with the indicated concentrations of insulin for 5 (PKCζ,) 15 (PKCδ)

or 30 min (PKCα,β) as indicated. The cells were solubilized and immunoprecipitated with

isoform-specific PKC antibodies. PKC activity was then assayed in the immunoprecipitates as

described under Materials and Methods. Bars represent the mean ± S.D. of duplicate

determinations in four independent experiments. A - For control, aliquots of the cell lysates

were blotted with isoform-specific PKC antibodies as indicated. Filters were revealed by ECL

and autoradiographed. The autoradiographs shown are representative of three immunoblots

for each of the indicated PKC isoforms.

Fig.9 PKCζ, glycogen synthase activities and GSK3 phosphorylation in L6hIR cells

overexpressing IRS-2. L6hIR cells were transiently transfected with the IRS-2 cDNA as

indicated and stimulated with 100 nM insulin. PKCζ (left panel) and glycogen synthase

activities (right panel) were then assayed as described in the legends to Fig.8 and 4,

respectively. Cell lysates were also blotted with Ser21phospho-GSK3α or Ser9phospho-

GSK3β antibodies (top right). For control, aliquots of the cell lysates were blotted with IRS-2

antibodies (top left). Filters were revealed by ECL and autoradiographed. Bars represent the

mean ± S.D. of duplicate determinations in three (PKCζ activity) and four (glycogen synthase

activity) independent experiments. The autoradiographs shown are representative of four

independent experiments.

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Fig.10 Effect of blocking of PKCζ activity on GSK3α and β function. B - L6hIR cells were

transiently transfected with the dominant negative PKCζ cDNA (PKCζ-DN), PKCζ antisense

(AS-ζ), or control antisense (S-ζ). The cell were then stimulated with 100 nM insulin as

indicated, solubilized and immunoprecipitated with GSK3α or GSK3β antibodies. GSK3

activity was then assayed in the immunoprecipitates as described under Materials and

Methods. A - For control, aliquots of the cell lysates were blotted with PKCζ or PKCδ

antibodies. Filters were revealed by ECL and autoradiographed. C - Alternatively, aliquots of

the cell lysates were precipitated with PKCz antibodies and precipitates assayed for PKC

activity as outlined in the legend to Fig.8. Bars represent the mean ± S.D. of duplicate

determinations in four (B) and three (C) independent experiments.

Fig.11 Effect of blocking of PKCζ or δ activities on Glycogen synthase function. L6hIR

cells were transiently transfected with the dominant negative PKCζ cDNA (PKCζ-DN),

PKCζ antisense (AS-ζ), or control antisense (S-ζ), or treated with 3 µM Rottlerin for 30 min,

as indicated. 100 nM insulin was then added followed by cell solubilization. Cell lysates were

assayed for glycogen synthase activity as described under Materials and Methods. Bars

represent the mean ± S.D. of duplicate determinations in four independent experiments.

Fig.12 PKCζ interaction with GSK3α and β. A - L6hIR cells were transiently transfected

with the dominant negative PKCζ cDNA (PKCζ-DN) or the empty vector cDNA, and

stimulated with 100 nM insulin for 5 min as indicated. The cells were then lysed and

immunoprecipitated with PKCζ antibodies, followed by blotting with GSK3α or GSK3β

antibodies. Filters were revealed by ECL and subjected to autoradiography. B,C - Purified

GSK3α and β were incubated with recombinant PKCζ in the presence of PKC activators and

[γ32P]ATP. The incubation mixtures were subjected to SDS-PAGE and autoradiography.

PKCζ and GSK3 were identified based on molecular size (and confirmed by immunoblot

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analysis of the bands). The auoradiographs shown are representative of three (A) and four

(B,C) independent experiments.

Fig.13 PKCζ and Akt/PKB effects on GSK3 activity. A - Purified GSK3α (0.1 µg) was

incubated with recombinant PKCζ (rPKCζ) and PKC activators, or with Akt/PKB

precipitated from either basal (IP-Akt(-)) or insulin-stimulated L6hIR cells (IP-Akt(+)), or with

recombinant active Akt/PKB (rAkt(active)) in reaction buffer (see Materials and Methods).

GSK3 activity was then assayed as described under Materials and Methods. Bars represent the

mean ± S.D. of duplicate determinations in three independent experiments. B - Aliquots of the

incubation mixtures were blotted with either pSer21GSK3α or pSer9GSK3β antibodies,

subjected to ECL and autoradiographed. C - L6hIR cells were transiently transfected with the

dominant negative PKCζ cDNA (PKCζ-DN), PKCζ antisense (AS-ζ), or control antisense

(S-ζ). The cells were subsequently stimulated with 100 nM insulin for 5 min, solubilized, and

blotted with either either pSer21GSK3α or pSer9GSK3β antibodies as indicated. Filters were

revealed by ECL. The autoradiographs shown are representative of three (B) and four (C)

independent experiments.

Fig.14 Effect of ML-9 on insulin activation of GSK3 and glycogen synthase. A - L6hIR

cells were incubated with 100 µM ML-9 for 10 min. During the last 5 min of incubation

insulin was added at 100 nM final concentration. The cells were solubilized and blotted with

pSer21GSK3α or pSer9GSK3β antibodies, as indicated. Filters were revealed by ECL and

autoradiogrphed. The autoradiographs shown are representative of three independent

experiments. B - Alternatively, the cell lysates were assayed for glycogen synthase activity as

described under Materials and Methods. Bars represent the mean ± S.D. of duplicate

determinations in four independent experiments.

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Gerolama Condorelli, Emmanuel Van Obberghen and Francesco BeguinotMaitan, Giovanni Vigliotta, Alessandra Trencia, Stefania Santopietro, Matilde Caruso,

Francesco Oriente, Pietro Formisano, Claudia Miele, Francesca Fiory, Maria AlessandraL6 cells

SRC="/math/zeta.gif" ALIGN="BASELINE" ALT="zeta "> activation by insulin in Insulin receptor substrate (IRS)-2 phosphorylation is necessary for PKC<IMG

published online July 31, 2001J. Biol. Chem. 

  10.1074/jbc.M104405200Access the most updated version of this article at doi:

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