The Adapter Protein ZIP Binds Grb14 and Regulates Its Inhibitory Action on Insulin Signaling by Recruiting Protein Kinase C{zeta}

Grb14 is a member of the Grb7 family of adapters and acts asa negative regulator of insulin-mediated signaling. Here wefound that the protein kinase C ${zeta}$ (PKC ${zeta}$ ) interacting protein, ZIP,interacted with Grb14. Coimmunoprecipitation experiments demonstratedthat ZIP bound to both Grb14 and PKC ${zeta}$ , thereby acting as a linkin the assembly of a PKC ${zeta}$ -ZIP-Grb14 heterotrimeric complex. Mappingstudies indicated that ZIP interacted through its ZZ zinc fingerdomain with the phosphorylated insulin receptor interactingregion (PIR) of Grb14. PKC ${zeta}$ phosphorylated Grb14 under in vitroconditions and in CHO-IR cells as demonstrated by in vivo labelingexperiments. Furthermore, Grb14 phosphorylation was increasedunder insulin stimulation, suggesting that the PKC ${zeta}$ -ZIP-Grb14complex is involved in insulin signaling. The PIR of Grb14,which also interacts with the catalytic domain of the insulinreceptor (IR) and inhibits its activity, was preferentiallyphosphorylated by PKC ${zeta}$ . Interestingly, the phosphorylation ofGrb14 by PKC ${zeta}$ increased its inhibitory effect on IR tyrosinekinase activity in vitro. The role of ZIP and Grb14 in insulinsignaling was further investigated in vivo in Xenopus laevisoocytes. In this model, ZIP potentiated the inhibitory actionof Grb14 on insulin-induced oocyte maturation. Importantly,this effect required the recruitment of PKC ${zeta}$ and the phosphorylationof Grb14, providing in vivo evidences for a regulation of Grb14-inhibitoryaction by ZIP and PKC ${zeta}$ . Together, these results suggest thatGrb14, ZIP, and PKC ${zeta}$ participate in a new feedback pathway ofinsulin signaling.

INTRODUCTION

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Introduction

Molecular adapters are proteins composed of the juxtapositionof various protein-protein or protein-lipid interacting domainsand devoided of enzymatic activity. These proteins are essentialcomponents of signal transduction pathways. The Grb7 familyof adapters, which comprises Grb7, Grb10, and Grb14, is implicatedin receptor tyrosine kinase (RTK) signaling (7, 41). Growingevidence is emerging for an inhibitory role of Grb14 and Grb10in insulin signaling. Grb14 is selectively expressed in insulin-sensitivetissues and upon insulin stimulation interacts in vivo withthe insulin receptor (IR). Moreover, the overexpression of Grb14in the CHO-IR cell line was shown to inhibit insulin-stimulatedtyrosine phosphorylation of specific proteins, like IRS-1, anddistal effects, like DNA and glycogen synthesis (23, 28). Despitecontroversial findings (46, 52, 71), overexpression studiesof Grb10 isoforms are also consistent with a negative role ininsulin signaling (34, 44, 48). The molecular analysis of theinteraction between the Grb7 family of proteins and IR recentlyled to clues on their inhibitory action. These proteins interactin an insulin-dependent manner with the activated tyrosine kinaseloop of the IR, and this interaction is mediated by their C-terminalregion, containing the phosphorylated IR interacting region(PIR, also known as BPS, between the pleckstrin homology [PH]domain and the Src 2 homology [SH2] domain) and SH2 domains(17, 21, 22, 27, 28, 34). Using in vitro tyrosine kinase assays,it was recently shown that the binding of the PIR inhibits IRtyrosine kinase activity (2, 67).

In addition to the PIR and SH2 domains, members of the Grb7family of proteins contain several conserved interacting regions,including a central PH domain, and an N-terminal proline-richmotif, which conforms to the consensus sequence of an SH3 bindingsite. These domains are binding sites for various proteins whichare potentially involved in RTK signaling. Many reports havedescribed interactions of the Grb proteins with various RTKs,but fewer studies have identified partners involved in post-receptorsignaling steps, most of them concerning Grb10 partners (fora review, see reference 20). Kinases, like the serine/threoninekinases MEK1 and Raf1 or the tyrosine kinases Src and Tec, andthe ubiquitin ligase Nedd4 have been shown to interact withthe SH2 domain of Grb10 (33, 38, 45, 50). It has also been proposedthat cAbl interacts with the proline-rich motif of Grb10 (17).To date, there is only one report of a nonreceptor Grb14 interactingprotein, a novel human tankyrase that has been identified inassociation with the N-terminal domain of Grb14. This tankyraseis an ankyrin repeat-containing protein which is likely to beinvolved in the subcellular localization of Grb14 (36).

To identify new downstream partners of Grb14, we performed atwo-hybrid screen of a rat liver cDNA library using the C-terminaldomain of Grb14 as a bait. In this study we showed that themolecular adapter ZIP (protein kinase C ${zeta}$ [PKC ${zeta}$ ] interacting protein),also known as p62 (55, 60), interacted with Grb14. ZIP linkedthe atypical PKC ${zeta}$ to Grb14 into a heterotrimeric complex in vivo.In addition, we demonstrated that PKC ${zeta}$ phosphorylated Grb14 andincreased its inhibitory action on IR catalytic activity invitro. Of functional relevance, these data correlated with theregulation by Grb14 and ZIP of insulin-induced maturation ofXenopus laevis oocyte. Together, these findings suggest thatGrb14, ZIP, and PKC ${zeta}$ are involved in a common down-regulationpathway of insulin signaling.

MATERIALS AND METHODS

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Materials and Methods

Reagents and antibodies. The following reagents were purchased from the indicated sources:oligonucleotides, culture media, and G418 (Geneticin) were fromLife Technologies; human recombinant PKC ${zeta}$ was from Upstate BiotechnologyInc.; cell-permeable myristoylated PKC ${zeta}$ pseudosubstrate inhibitor(Myr-SIYRRGARRWKRL) was from Calbiochem; hygromycin and monoclonalantihemagglutinin (anti-HA) antibodies were from Roche MolecularBiochemicals; monoclonal anti-myc and anti-V5 antibodies werefrom Invitrogen; monoclonal anti-ZIP (p62) antibodies were fromTransduction Laboratories, Inc.; polyclonal anti-PKC ${zeta}$ and anti-extracellularsignal-regulated kinase 2 (anti-ERK2) antibodies were from SantaCruz; and poly Glu-Tyr (4:1) and all chemicals were from SigmaFrance. Polyclonal anti-Grb14 antibodies were described previously(28). Polyclonal anti-ZIP antibodies were raised against a glutathioneS-transferase (GST)-ZIP ${Delta}$ AID fusion protein and purified on proteinA-agarose before use.

Yeast two-hybrid screen. A yeast two-hybrid screen was performed in the Saccharomycescerevisiae strain L40 using on, one hand, pFBL23-Grb14, encodingthe C-terminal domain of Grb14 (amino acids 346 to 538) in N-terminalfusion with the LexA binding domain and, on the other hand,an oligo(dT)-primed cDNA library from a rat liver, cloned infusion with the Gal4 activation domain in the pGAD3S2X plasmid,as previously described (28). The plasmids of the library producingyeast colonies of a His⁺ LacZ⁺ phenotype were isolated, andthe specificity of association of their products with Grb14was tested using pLex-lamin as a negative control. The cDNAinserts of these positive plasmids were sequenced.

5' RACE and cDNA cloning. pGAD-3'ZIP fragment, encompassing amino acids 104 to 440 ofrat ZIP, was obtained by the two hybrid screen. To clone the5' end of the ZIP cDNA, the 5' rapid amplification of cDNA ends(5' RACE) technique was used on a rat liver Marathon Ready premadecDNA library (Clontech), with the Advantage cDNA PCR kit (Clontech).We used a primer corresponding to the 5' sequence of ZIP, upstreamfrom the ATG, according to the published sequence of rat ZIP(GenBank accession no.Y08355) and an antisense primer designedin the cloned insert. We obtained a 940-bp fragment correspondingto the 5' end of the cDNA. This fragment was subcloned by TAcloning into the pTAdv vector (Clontech). An EcoRI/BamHI fragment(750 bp long) was then excised from the pTAdv-5'ZIP plasmidand ligated to pGAD-3'ZIP digested by EcoRI and BamHI, generatingpGAD-ZIP (full length).

Plasmid constructions. ZIP ${alpha}$ , ZIPß, ZIP ${Delta}$ AID+ZZ ( ${Delta}$ 1-163), ZIP ${Delta}$ AID ( ${Delta}$ 1-103) ZIPZZ(104-163), ZIPAID+ZZ (1-163) were amplified from the pGAD-ZIPplasmid using primers to introduce an EcoRI site in the 5' endand an XhoI site in the 3' end and then cloned in frame in apGEX4T2 vector (Amersham Pharmacia Biotech) to produce GST-ZIPfusion proteins. ZIP ${alpha}$ and ZIPß were also cloned inpcDNA6/V5-HisC (Invitrogen) for expression of a V5 epitope-taggedversion. All constructs were verified by DNA sequence analysis.pECE-myc-Grb14, pGEX-Grb7, pGEX-Grb10, and all pGEX-Grb14 constructswere described previously (2, 28).

Cell culture and preparation of lysates. Chinese hamster ovary cells stably overexpressing IR (CHO-IR)or IR and Grb14 (CHO-IR-Grb14) were grown in Ham F-12 mediumsupplemented with 1 mM glutamine, penicillin (100 U/ml), streptomycin(100 µg/ml), and 10% fetal bovine serum, in the presenceof G418 (75 µg/ml) (CHO-IR) and with the addition of hygromycin(100 µg/ml) (CHO-IR-Grb14) (28). COS cells were grownin Dulbecco's modified Eagle's medium, supplemented with 1 mMglutamine, penicillin (100 U/ml), streptomycin (100 µg/ml),and 10% fetal bovine serum. Confluent cells were transientlytransfected using Lipofectamine (Invitrogen) as recommendedby the manufacturer. Cells were serum starved for 24 h and stimulatedor not with insulin (10^-7 M) for 10 min at 37°C. They werethen solubilized at 4°C in lysis buffer (20 mM Tris-HCl[pH 7.5], 150 mM NaCl, 5 mM EDTA, 30 mM sodium pyrophosphate,50 mM NaF, 1% Triton X-100, pepstatin [1 µg/ml], leupeptin[2 µg/ml], aprotinin [5 µg/ml], 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate) with gentle rotation at4°C for 30 min. Cell lysates were then cleared by a 15,000x g centrifugation at 4°C for 15 min.

Production of GST fusion proteins. Purified GST fusion proteins were produced as described previously(29). Fusion proteins were quantified by Coomassie blue stainingafter sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis. For in vitro phosphorylation experimentsand Xenopus oocyte microinjections, the fusion proteins wereeluted from the glutathione-Sepharose beads with 50 mM Tris-HCl,pH 8, containing 10 mM reduced glutathione, and quantified asdescribed above.

In vitro interaction assays. Cell lysates (250 µg) were incubated overnight at 4°Cwith 3 µg of immobilized GST fusion proteins. For in vitrointeraction with PKC ${zeta}$ , 2.5 ng of human recombinant PKC ${zeta}$ was incubatedduring 1 h at 4°C in 500 µl of lysis buffer with 1.5µg of immobilized GST fusion proteins. After extensivewashing, bound proteins were eluted by heating in SDS samplebuffer, separated by SDS-PAGE, and transferred to nitrocellulose.We verified by Ponceau red staining of the nitrocellulose membranesthat equal amounts of GST fusions were used. Membranes werethen subjected to immunodetection with the indicated antibodies.Immunoreactive bands were revealed using an ECL detection kit(Amersham Pharmacia Biotech).

Immunoprecipitation and Western blot analysis. For coimmunoprecipitation experiments, cells were transientlytransfected with the indicated expression plasmids. Beginningat 48 h after transfection, cells were serum starved for 24h and stimulated or not with insulin (10^-7 M) for 10 min at37°C. Cell lysates were prepared as described above. Aftera preclearing step, 500 µg of cell lysates was incubatedovernight at 4°C with the indicated antibodies in the presenceof 100 µl of protein A-agarose. The immunoprecipitateswere washed three times in lysis buffer, subjected to SDS-PAGE,and immunodetected with the indicated antibodies. Immunoreactivebands were revealed as explained above.

For in vivo studies in rats, liver proteins were extracted inlysis buffer, and then lysates were solubilized with gentlerotation for 90 min at 4°C and cleared by a 15,000 x g centrifugationat 4°C for 15 min. Protein concentrations were determinedusing a bicinchoninic acid protein assay (Pierce). Immunoprecipitationswere performed as described above.

In vitro phosphorylation of Grb14 by PKC ${zeta}$ . The PKC ${zeta}$ kinase assays were performed essentially as recommendedby the manufacturer (Upstate Biotechnology Inc.). Human recombinantPKC ${zeta}$ (50 ng) was preincubated with the indicated amounts of elutedGST fusion proteins during 10 min at room temperature in assaydilution buffer II (20 mM MOPS [pH 7.2], 25 mM ß-glycerophosphate,1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM calciumchloride). The kinase reaction (final reaction mixture volume,60 µl) was initiated by the addition of 5 µg ofphosphatidylserine, 7.5 mM MgCl₂, 50 µM ATP, and 5µCiof [ ${gamma}$ -³²P]ATP. After a 30-min incubation at 30°C, the reactionwas stopped by the addition of Laemmli sample buffer and thesamples were boiled for 5 min. Samples were then subjected toSDS-10% PAGE, and the bands corresponding to phosphorylatedproteins were detected by autoradiography. For in vitro IR tyrosinekinase assays with GST-PIR-SH2, and for microinjection of phosphorylatedGST-PIR into Xenopus oocytes, 2 µg of GST fusion proteinswas incubated with 50 ng of PKC ${zeta}$ as described above. Variousamounts of GST fusion proteins were obtained by dilution withassay dilution buffer II.

Metabolic labeling. Confluent CHO-IR cells were transiently transfected with myc-Grb14,HA-PKC ${zeta}$ , and pcDNA6 (empty vector) as indicated. Beginning at24 h after transfection, cells were serum starved for 16 h.Cells were labeled for 3 h in the same medium containing 100µCi/ml of [³²P]orthophosphate (Amersham Pharmacia Biotech)and then stimulated or not with insulin (10^-7M) for 10 min at37°C. When indicated, 20 µM PKC ${zeta}$ pseudosubstrate inhibitorwas added 30 min before insulin stimulation. Cells were washedtwice in KRBH buffer, pH 7.4 (107 mM NaCl, 5 mM KCl, 3 mM CaCl₂,1 mM MgSO₄, 20 mM HEPES, 10 mM glucose, 1% bovine serum albumin,7 mM NaHCO₃) and scraped into lysis buffer. Lysates were clearedby a 15,000 x g centrifugation at 4°C for 15 min. The supernatantfractions were immunoprecipitated with anti-Grb14 polyclonalantibodies. Immunocomplexes were washed three times with lysisbuffer, boiled in Laemmli sample buffer, separated by SDS-10%PAGE, and transferred to nitrocellulose. Phosphorylated Grb14was detected with a PhosphorImager (Molecular Dynamics) andquantified (ImageQuant). The nitrocellulose membranes were thenimmunodetected using anti-myc antibodies.

IR tyrosine kinase activity in vitro assays. IRs were partially purified by affinity chromatography on wheatgerm agglutinin (WGA), essentially as described previously (2).WGA-purified receptors were incubated for 1 h at room temperaturein the presence or in the absence of 100 nM insulin, in 30 mMHEPES buffer (pH 7.6), supplemented with 30 mM NaCl, 0.1% TritonX-100, and 0.03% bovine serum albumin. The autophosphorylationof receptors was initiated by the addition of phosphorylationbuffer (8 mM MgCl₂, 4 mM MnCl₂, 20 µM ATP, and 5 µCiof [ ${gamma}$ -³²P]ATP per sample) for 30 min at room temperature, inthe presence of variable amounts of GST-PIR-SH2 of Grb14, previouslyphosphorylated by PKC ${zeta}$ or not. The tyrosine kinase assay wasperformed using 15 µg of the synthetic substrate polyGlu-Tyr (4:1). After 30 min the reaction was stopped by spottingthe mixture onto Whatman ET31 filter paper squares (2 by 2 cm),which were treated as described previously (2). Results wereexpressed as a percentage of the maximal insulin-stimulatedkinase activity measured in the absence of added fusion proteins.

Xenopus oocyte studies. After anesthesia with MS 222 (1 g/liter; Sandoz), Xenopus laevisovarian fragments were surgically removed and placed in modifiedOR2 medium lacking potassium (82.5 mM NaCl, 1 mM MgCl₂, 1 mMCaCl₂, 1 mM Na₂HPO₄, 5 mM HEPES, adjusted to pH 7.4), supplementedwith streptomycin-penicillin (each at 50 µg/ml; Eurobio),tetracycline (50 µg/ml; Sigma), sodium pyruvate (225 µg/ml;Sigma), and soybean trypsin inhibitor (30 µg/ml; Sigma).Stage VI oocytes were harvested by using 1-h treatment withcollagenase A (1 mg/ml; Boehringer Mannheim). Complete defolliculationof the oocytes was achieved by manual dissection. The oocyteswere kept at 19°C in the modified OR2 medium. Microinjectionsof eluted GST fusion proteins were performed in the equatorialregion of the oocyte. Germinal vesicle breakdown (GVBD) wasdetermined by the appearance of a white spot at the center ofthe animal pole. For ERK2 phosphorylation analysis, oocyteswere lysed in ice-cold homogenization buffer (60 mM ß-glycerophosphate,15 mM nitrophenylphosphate, 15 mM EGTA, 15 mM MgCl₂, 2 mM dithiothreitol,1 mM sodium orthovanadate, 1 mM NaF, leupeptin [10 µg/ml],aprotinin [10 µg/ml], soybean trypsin inhibitor [10 µg/ml],1 mM phenylmethylsulfonyl fluoride, 100 µM benzamidine,25 mM MOPS [pH 7.2]). Electrophoresis was performed on 15% modifiedpolyacrylamide gels (30% acrylamide and 0.2% bisacrylamide).Western blot analysis was performed as described above.

RESULTS

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Results

Cloning of ZIP by interaction with Grb14 in a yeast two-hybrid screen. A rat liver cDNA library was screened using the C-terminal domainof Grb14, including the PIR and SH2 domains as a bait (aminoacids 346 to 538) (Fig. 1A). The rat used for the library constructionhad been starved for 48 h and refed with a high-carbohydratediet, to favor the identification of genes involved in insulin-stimulatedmetabolism. From 2 x 10⁶ tested colonies, eight clones containedplasmids encoding proteins interacting with Grb14, and not witha nonspecific control like lamin. Sequence analysis of the cDNAsinserted in these plasmids revealed that all encoded the sameprotein, although they can be divided into two groups, differingby a deletion of 114 bp in the 3' end. The longest insert was1.6 kb and encoded the last 337 amino acids of the protein ZIP(amino acids 104 to 440). ZIP was previously isolated by itsinteraction with the atypical PKC ${zeta}$ (55, 60). ZIP also interactswith several proteins like p56^lck (26), RIP (62), TRAF6 (61),COUP-TFII (40), and Kvß2 (18). The full-length ratZIP cDNAs were cloned using the 5'-RACE technique. The full-length440-amino-acid ZIP protein was called ZIP ${alpha}$ . The shorter form,encoded by the cDNA with 114 bp deleted, carried a deletionof amino acids 349 to 387 and was called ZIPß. Thisvariant is different from the previously reported isoform ZIP2(deletion of amino acids 222 to 248) (18). ZIP is a molecularadapter, characterized by the succession of several conservedprotein domains (Fig. 1B): an N-terminal atypical PKC-interactingdomain (AID), a cysteine-rich sequence called ZZ zinc fingerdomain, two central PEST domains, which are likely to be implicatedin protein stability, and a C-terminal ubiquitin-associateddomain potentially involved in ubiquitination-mediated proteindegradation (55, 61, 70). The ZIPß isoform lackedthe second PEST domain.

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FIG. 1. Schematic representation of Grb14 and ZIP proteins. (A) Grb14. Shown are the proline-rich motif (PP); the PH domain; the PIR (28), also called BPS, between the PH and SH2 domain (22); and the SH2 domain. (B) ZIP and various ZIP constructs. The arrow indicates the length of the ZIP protein isolated in the two-hybrid screen. Shown are the AID (47), ZZ, PEST sequences (PEST2 is lacking in the ZIPß isoform), and ubiquitin-associated (UBA) domain (70).

ZIP forms a heteromultimeric complex with Grb14 and PKC ${zeta}$ . The Grb14-ZIP interaction observed in yeast was first confirmedin vitro using GST pull down assays. Immobilized GST-ZIP ${alpha}$ andGST-ZIPß were incubated with CHO-IR-Grb14 cell lysates,and retained proteins were analyzed on anti-Grb14 immunoblots.As shown in Fig. 2A, Grb14 was similarly retained by GST-ZIP ${alpha}$ and GST-ZIPß. GST alone did not bind to Grb14. Basedon the observation that PIR and SH2 domains are conserved betweenthe members of Grb7 family (8, 28), we compared their respectivebinding abilities to that of ZIP ${alpha}$ . A series of constructs ofeach Grb7 family protein fused to GST were immobilized on glutathione-Sepharoseand used to pull down ZIP ${alpha}$ from lysates of transiently transfectedCOS cells. As shown in Fig. 2B, Grb10 and Grb14 bound to ZIP ${alpha}$ ,whereas no interaction was detected with Grb7.

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FIG. 2. Interaction of Grb14 with ZIP in vitro. (A) ZIP isoforms expressed as GST-fusion proteins or GST control were immobilized on glutathione-Sepharose beads and incubated with lysates of CHO-IR-Grb14 cells. Bound proteins were immunodetected with anti-Grb14 polyclonal antibodies. (B) Grb7 family members expressed as immobilized GST-fusion proteins were incubated with lysates of COS cells transiently transfected with V5 tagged ZIP ${alpha}$ . Bound proteins were detected by immunodetection with anti-V5 monoclonal antibodies. These blots are representative of 3 different experiments.

To verify that the interaction between Grb14 and ZIP also occursin intact mammalian cells, CHO-IR cells were transiently transfectedwith myc-Grb14 and V5-ZIP ${alpha}$ . At 48 h after transfection, cellswere stimulated or not with insulin, and cell lysates were immunoprecipitatedwith anti-Grb14 polyclonal antibodies. The anti-Grb14 immunoprecipitateswere analyzed with anti-V5 antibodies. As shown in Fig. 3A (upperblots, lanes 4 and 5), V5-ZIP ${alpha}$ coprecipitated with myc-Grb14under basal conditions as well as after insulin stimulation.Similar results were obtained when using the V5-ZIPßexpression plasmid (data not shown).

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FIG. 3. ZIP forms a ternary complex with Grb14 and PKC ${zeta}$ in vivo. (A) CHO-IR cells were transiently transfected as indicated with 1 µg of either pECE myc-Grb14, pcDNA6 V5-ZIP ${alpha}$ , or pcDNA3 HA-PKC ${zeta}$ . pcDNA6 empty plasmid was used to normalize total DNA amount to 3 µg. At 48 h after transfection, cells were stimulated or not with 10^-7 M insulin. Cell extracts were immunoprecipitated with polyclonal anti-Grb14 antibodies. Control immunoprecipitations using rabbit preimmune serum were simultaneously performed (PI). The immunoprecipitates were separated by SDS-PAGE and immunoblotted with monoclonal anti-HA, anti-V5, or anti-myc antibodies. Lysates of the same cells were separated by SDS-PAGE and immunoblotted with the same antibodies to confirm equal expressions levels of proteins. (B) Grb14 does not interact with PKC ${zeta}$ in vitro. ZIP ${alpha}$ and Grb14 expressed as GST fusion proteins were incubated with human recombinant PKC ${zeta}$ . Bound PKC ${zeta}$ was detected by Western blotting using anti-PKC ${zeta}$ antibodies. (C) Lysates from COS cells transiently transfected with myc-Grb14 were immunoprecipitated with polyclonal anti-Grb14 antibodies. Control immunoprecipitation using rabbit preimmune serum was simultaneously performed. The immunoprecipitates were separated by SDS-PAGE and immunoblotted as indicated with monoclonal anti-ZIP antibodies, anti-myc antibodies, or polyclonal anti-PKC ${zeta}$ antibodies. Lysates of the same cells were analyzed in parallel. (D) Rat liver extracts were immunoprecipitated with polyclonal anti-Grb14 antibodies cross-linked to protein A-agarose. Control immunoprecipitation using rabbit preimmune serum was simultaneously performed. Immunoprecipitates were analyzed by sequential immunodetection using polyclonal anti-ZIP and anti-PKC ${zeta}$ antibodies. In this figure the blots are representative of two or three independent experiments, and lysates and preimmune controls were analyzed on the same gels as experimental samples.

Previous studies have shown that ZIP interacts with PKC ${zeta}$ andbehaves as a link between PKC ${zeta}$ and other proteins, such as RIP(62) or Kvß2 (18), in ternary complexes. Therefore,we next investigated whether ZIP could simultaneously interactwith Grb14 and PKC ${zeta}$ . CHO-IR cells were transfected with myc-Grb14,V5-ZIP ${alpha}$ , and HA-PKC ${zeta}$ , and cell extracts were immunoprecipitatedwith anti-Grb14 antibodies. The immunoprecipitates were thenanalyzed with either anti-V5 or anti-HA antibodies. The resultsof Fig. 3A (lanes 6 and 7) indicate that V5-ZIP ${alpha}$ and HA-PKC ${zeta}$ coprecipitatedwith myc-Grb14, in the basal state as well as after insulinstimulation. To further demonstrate that ZIP is the link thatconnects PKC ${zeta}$ to Grb14, CHO-IR cells were transfected with onlymyc-Grb14 and HA-PKC ${zeta}$ . Interestingly, HA-PKC ${zeta}$ was immunoprecipitatedwith the myc-Grb14 antibodies. However, the amount of PKC ${zeta}$ immunoprecipitatedwas lower than in the presence of coexpressed V5-ZIP ${alpha}$ (middleblots [compare lanes 8 and 9 to lanes 6 and 7]). As ZIP is expressedin CHO cells (data not shown), endogenous ZIP was likely tomediate the interaction between Grb14 and PKC ${zeta}$ . To verify thishypothesis, we tested direct in vitro interaction between PKC ${zeta}$ and Grb14. Immobilized GST-ZIP ${alpha}$ and GST-Grb14 were incubatedwith recombinant PKC ${zeta}$ , and retained proteins were analyzed onanti-PKC ${zeta}$ immunoblots. As shown in Fig. 3B, PKC ${zeta}$ was retainedby GST-ZIP ${alpha}$ as expected, whereas it did not interact with GST-Grb14or GST alone. All together, these results indicate that ZIPforms a bridge between Grb14 and PKC ${zeta}$ .

Next, we attempted to confirm that these interactions also occurunder more-physiological conditions. Since Grb14 was not expressedin various cell lines, like CHO or COS cells (data not shown),we investigated whether endogenous ZIP and PKC ${zeta}$ can form a complexwith ectopically expressed Grb14. COS cells were transientlytransfected with myc-Grb14. Under these conditions, the levelof expression of Grb14 protein was about fourfold higher thanthat in rat liver (data not shown). Cell extracts were immunoprecipitatedwith anti-Grb14 antibodies, and the immunoprecipitates wereanalyzed with either monoclonal anti-ZIP or polyclonal anti-PKC ${zeta}$ antibodies. As reported in Fig. 3C, endogenous ZIP and PKC ${zeta}$ coprecipitatedwith myc-Grb14, whereas no interaction was detected with nonspecificrabbit immunoglobulin G.

Grb14 is specifically expressed in insulin-sensitive tissues(28). Then, we used rat liver extracts to further demonstratethe heterotrimeric complex formation in vivo, in a physiologicaltarget of insulin. In agreement with the data obtained withtransfected cell lines, we were able to detect a ternary complexbetween endogenous Grb14, ZIP, and PKC ${zeta}$ in a coimmunoprecipitationexperiment using anti-Grb14 antibodies (Fig. 3D). This resultsuggests that the constitution of the heterotrimeric complexmay be physiologically relevant.

Mapping of regions involved in the Grb14-ZIP association. In order to determine which region of ZIP is required for theinteraction with Grb14, a series of ZIP constructs fused toGST (Fig. 1B) was used to pull down Grb14 from lysates of CHO-IR-Grb14cells. As shown in Fig. 4A, deletion of the N terminus of ZIP,containing the AID and ZZ domains, suppressed Grb14 interaction(lanes 1, 2, and 6). The complementary construct, amino acids1 to 163 of ZIP, retained Grb14, but to a lower extent thanwild-type (WT) ZIP (compare lanes 2 and 3). This suggests thatthe presence of the following domains in C terminus of the ZZdomain is required for a correct folding of the protein, andthus for a full binding activity. The ${Delta}$ AID construct, containingthe ZZ domain and the C terminus of the protein, bound Grb14,but also with a lower affinity than WT ZIP (compare lanes 2and 5). This decreased binding could be explained by a roleof the AID domain, ZIP amino acids 1 to 103, in Grb14 binding.However, this is unlikely since (i) a similar amount of Grb14was retained by the AID-ZZ and the ZZ domains (lanes 3 and 4);(ii) the clones isolated in the two-hybrid screen did not containthe AID domain (Fig. 1B), suggesting that it is dispensablefor the ZIP-Grb14 interaction in yeast; and (iii) similar amountsof WT ZIP and ZIP ${Delta}$ AID were retained in anti-Grb14 immunoprecipitationexperiments performed in mammalian cells (data not shown). Thus,more probably, the presence of the GST in N terminus of theZIP ${Delta}$ AID construct had altered the protein folding and partiallymasked the ZZ interaction domain. Although we cannot excludefrom these experiments the possibility that other domains ofZIP participate in the interaction with Grb14, these resultsare in favor of a role of the ZZ domain (amino acids 104 to163) in Grb14 binding.

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FIG. 4. Mapping of domains involved in the Grb14-ZIP interaction. (A) ZIP ${alpha}$ and various ZIP constructs expressed as GST fusion proteins were incubated with lysates of CHO-IR-Grb14 cells. Bound proteins were immunodetected with anti-Grb14 antibodies. (B) Various Grb14 constructs expressed as GST fusion proteins were incubated with lysates of COS cells transiently transfected with V5-ZIP ${alpha}$ . Bound proteins were immunodetected using anti-V5 antibodies. Ponceau red staining of the proteins after transfer confirmed that equivalent amounts of GST fusions were loaded. These blots are representative of three different experiments.

By the same approach, using a series of Grb14 deletion constructsfused to GST, we investigated the role of the different domainsof Grb14 in its binding to ZIP. Figure 4B clearly shows thatthe strongest binding was detected with the PIR domain alone.By contrast, the SH2 domain was unable to bind to ZIP ${alpha}$ . The PIR-SH2domain and the full-length Grb14 bound to ZIP to a lower extentthan did the PIR alone. This decreased binding could indicatethat the presence of the other domains modifies the conformationof the PIR and reduces its binding affinity to ZIP. Collectively,these results suggest that the Grb14-ZIP binding is mediatedby the PIR-ZZ interaction.

PKC ${zeta}$ phosphorylates Grb14 in vitro and in intact cells. Previous studies reported that ZIP allows PKC ${zeta}$ to be associatedwith specific effectors in signaling complexes and to phosphorylatethem (18). Thus, to determine whether PKC ${zeta}$ was able to phosphorylateGrb14, we performed in vitro phosphorylation assays using humanrecombinant PKC ${zeta}$ and GST-Grb14 fusion protein. Incubation ofPKC ${zeta}$ with increasing amounts of GST-Grb14 resulted in an increasedphosphorylation of GST-Grb14, whereas control GST was not phosphorylated(Fig. 5, lanes 3, 4, and 7). The addition of GST-ZIP ${alpha}$ or GST-ZIPßto the reaction did not modify the level of Grb14 phosphorylation(data not shown), suggesting that under in vitro conditions,ZIP was dispensable for Grb14 phosphorylation by PKC ${zeta}$ . To determineif a particular domain of Grb14 was preferentially phosphorylatedby PKC ${zeta}$ , similar experiments were performed using various GST-Grb14constructs. The C-terminal domain of Grb14, including the PIRand SH2 domains (amino acids 346 to 538), was more intensivelyphosphorylated than the N-terminal domain (amino acids 1 to346) (Fig. 5, lanes 5 and 6). Interestingly, the PIR was a muchbetter substrate for PKC ${zeta}$ than the SH2 domain (Fig. 5, lanes8 to 10).

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FIG. 5. PKC ${zeta}$ phosphorylates Grb14 in vitro. Phosphorylation assays were performed in vitro using human recombinant PKC ${zeta}$ as described in Material and Methods. GST fusion proteins were used as substrate as indicated. Phosphorylated proteins were subjected to SDS-PAGE and analyzed by autoradiography. The asterisk indicates autophosphorylated PKC ${zeta}$ . These blots are representative of four different experiments.

In order to assess the phosphorylation state of Grb14 in intactcells, we performed in vivo metabolic labeling experiments.First, CHO-IR cells were transiently transfected with myc-Grb14,labeled with [³²P]orthophosphate, and stimulated or not withinsulin for 10 min. Consistent with previous results (8, 58),Grb14 was phosphorylated under basal conditions. Treatment ofthe cells with insulin induced a 60% increase in Grb14 levelof phosphorylation (Fig. 6). The addition of cell-permeablemyristoylated PKC ${zeta}$ pseudosubstrate (Myr-SIYRRGARRWRKL), whichspecifically inhibits PKC ${zeta}$ enzyme (65), decreased insulin-stimulatedGrb14 phosphorylation, restoring phosphorylation to the levelobserved in the absence of insulin. Cotransfection of CHO-IRcells with HA-PKC ${zeta}$ resulted in a 60% increase in insulin-stimulatedGrb14 phosphorylation. By contrast, in the absence of insulinstimulation, the overexpression of PKC ${zeta}$ did not increase Grb14phosphorylation (data not shown). Together, these results supportthe hypothesis that PKC ${zeta}$ may account for the in vivo insulin-inducedGrb14 phosphorylation.

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FIG. 6. Grb14 phosphorylation in intact cells. CHO-IR cells transiently transfected with myc-Grb14 and labeled with [³²P]orthophosphate were stimulated (+) or not (-) with insulin as indicated. Cells were either cotransfected with HA-PKC ${zeta}$ or empty vector (right part) or treated 30 min with 20 µM of myristoylated PKC ${zeta}$ (Myr-SIYRRGARRWRKL) pseudosubstrate prior to insulin stimulation (left part). Cell lysates were immunoprecipitated with anti-Grb14 antibodies, separated by SDS-PAGE, transferred to nitrocellulose membrane, and quantified with a PhosphorImager (upper blot). Anti-myc immunoblots were then performed to confirm that equal amounts of Grb14 were immunoprecipitated (lower blot). Results are expressed as a percentage of their respective control, in the absence of insulin (left part) or in the absence of HA-PKC ${zeta}$ (right part), and represent the means + standard errors of the means (error bars) of three (right part) or four (left part) independent experiments.

The phosphorylation of Grb14 by PKC ${zeta}$ increases its inhibitory effect on in vitro IR tyrosine kinase activity. The PIR of Grb14 mediates both the binding to the IR and theinhibitory action of the protein on the catalytic activity ofthe receptor (2, 28). As the PIR was the main domain of Grb14phosphorylated by PKC ${zeta}$ , we hypothesized that this phosphorylationcould modify the inhibitory action of Grb14 on IR catalyticactivity. IR tyrosine kinase activity was then investigatedin vitro in the presence of increasing amounts of GST-PIR-SH2,which was or was not previously phosphorylated by PKC ${zeta}$ . As shownin Fig. 7, the dose-response curve of IR kinase inhibition byphosphorylated GST-PIR-SH2 was shifted to the left comparedto that of inhibition by the nonphosphorylated protein, reflectingan increased sensitivity of the IR to its inhibitory effect.Fifty percent inhibition of IR tyrosine kinase activity wasobserved for 40 ng of phosphorylated GST-PIR-SH2 versus 160ng for the nonphosphorylated form. When performing the IR kinaseassay, PKC ${zeta}$ was added together with phosphorylated GST-PIR-SH2.As serine-threonine phosphorylation of IR has been describedto inhibit IR tyrosine kinase activity (4, 68, 69), we testedthe ability of PKC ${zeta}$ to modify the IR catalytic activity underthe same conditions. However, when added alone, the same amountsof PKC ${zeta}$ did not modify IR tyrosine kinase activity. The displacementof the dose-response curve was then attributable to the phosphorylationof the PIR-SH2 domain. Thus, these results clearly show thatphosphorylation of Grb14 by PKC ${zeta}$ increases its inhibitory actionon IR catalytic activity.

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FIG. 7. The phosphorylation of PIR-SH2 of Grb14 increases its inhibitory effect on IR catalytic activity. Tyrosine kinase activity of WGA-purified IR was measured using a synthetic substrate (poly Glu-Tyr 4:1), in the presence of increasing amounts of GST-PIR-SH2 previously phosphorylated or not by PKC ${zeta}$ , or in the presence of PKC ${zeta}$ alone. Results are expressed as a percentage of the maximal insulin effect measured in the absence of GST fusion proteins, and are the means + standard errors of the means of three to five different experiments.

ZIP potentiates the Grb14 inhibitory action on insulin-induced reinitiation of meiosis in X. laevis oocytes. Xenopus oocyte, physiologically arrested at the G₂ stage ofthe first meiotic prophase, is a suitable system for investigatingthe functional roles of signaling molecules initiated by severalextracellular inducers like progesterone or insulin (15, 37,42). The binding of these hormones to their receptors stimulatesthe entry of oocytes into the M phase, leading to a GVBD, anevent that can be easily monitored and used as an indicatorof reinitiation of meiosis. The effect of Grb14 and its domainson insulin-induced oocyte maturation was studied by microinjectionof GST-fusion proteins 1 h prior to stimulation with insulin(10^-6 M). As shown in Fig. 8A, microinjection of 100 ng of GST-Grb14totally blocked the maturation induced by insulin. Injectionof the same amount of GST had no effect on insulin-induced GVBD.In addition, this inhibitory action of Grb14 was specific forthe insulin transduction pathway, since it was not observedwhen the oocytes were stimulated with progesterone (data notshown). Interestingly, the PIR-SH2 and the PIR-GST fusions weresufficient to inhibit the reinitiation of meiosis, whereas theGST-SH2 fusion had no effect. The microinjection of 100 ng ofGST-ZIP ${alpha}$ , (or GST-ZIPß [data not shown]) did not alterthe insulin-induced reinitiation of oocyte meiosis.

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FIG. 8. Grb14 inhibits insulin-induced X. laevis oocyte maturation. Oocytes were microinjected with 100 ng of the indicated GST fusion proteins 1 h before insulin stimulation and analyzed 20 h later for the appearance of GVBD. (A) Effect of the injection of various GST fusions on the insulin induction of oocyte maturation. Results are expressed as a percentage of GVBD. Data represent the means + standard errors of the means (error bars) of two to three animals, each concerning 20 oocytes. (B) Western blot analysis of oocyte lysates using anti-ERK2 antibodies. The blot is representative of three different experiments.

Numerous studies have shown the importance of p21^ras-mitogen-activatedprotein (MAP) kinase cascade in insulin-induced GVBD (19, 54),and MAP kinase activation appears to be necessary and sufficientto induce oocyte maturation (19). Thus, to further investigatethe molecular mechanism of Grb14 inhibitory action in X. laevisoocytes, we studied its effect on MAP kinase activation. Insulin-inducedERK2 phosphorylation was detected by immunoblot analysis usinganti-ERK2 antibodies (Fig. 8B). A shift to a higher electrophoreticmobility of the 42-kDa protein, indicative of the presence ofERK2 phosphorylated form, was observed in oocytes stimulatedwith insulin but did not occur in oocytes previously microinjectedwith GST-Grb14. As expected, GST-ZIP ${alpha}$ and GST did not alter ERKphosphorylation. These data support the notion that Grb14 inhibitsthe IR catalytic activity and subsequent activation of MAP kinaseduring insulin stimulation (2).

The study of the effect of decreasing amounts of Grb14 indicatedthat at least 50 ng of GST-Grb14 was required to inhibit insulin-inducedGVBD (Fig. 9). Coinjection of 100 ng of GST-ZIP ${alpha}$ with low amountsof GST-Grb14, which were unable to inhibit the action of insulin,restored the inhibition of insulin-induced GVBD (Fig. 9). Similarresults were obtained when using ZIPß (data not shown).Thus, these results suggest that ZIP potentiates the inhibitoryaction of Grb14 on oocyte maturation.

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FIG. 9. ZIP potentiates Grb14 inhibition of insulin-induced oocyte maturation. Oocytes were microinjected with decreasing amounts of GST-Grb14 as indicated, together with (filled bars) or without (grey bars) 100 ng of GST-ZIP ${alpha}$ . Oocyte maturation in response to insulin was monitored as in Fig. 8. Data are the means + standard errors of the means (error bars) of three animals, each concerning 20 oocytes.

ZIP must interact with PKC ${zeta}$ to potentiate the action of Grb14. To further explore the mechanism by which ZIP regulates theaction of Grb14, we used ZIP deletion constructs expressed asGST-fusion proteins: ZIP ${Delta}$ AID+ZZ, from which both Grb14 and PKC ${zeta}$ interacting domains were deleted, and ZIP ${Delta}$ AID, which could notbind to PKC ${zeta}$ but retained its Grb14 binding ability. As shownpreviously, coinjection of ZIP ${alpha}$ with 10 ng of GST-Grb14 inhibitedinsulin-induced GVBD. By contrast, the coinjection of ZIP ${Delta}$ AID+ZZdid not allow the blocking of insulin-induced reinitiation ofmeiosis (Fig. 10). Interestingly, ZIP ${Delta}$ AID was also unable topotentiate the inhibitory effect of Grb14. These data suggestthat the ZIP-Grb14 interaction was not sufficient to mediatethe functional action of ZIP, and that ZIP must associate withboth Grb14 and PKC ${zeta}$ to potentiate Grb14 inhibitory action onoocyte Xenopus maturation. Similar results were obtained whenusing the PIR of Grb14 instead of full-length protein (Fig.10), whereas the SH2 domain had no effect (data not shown).Ten nanograms of GST-PIR was not sufficient to block the insulin-inducedreinitiation of oocyte meiosis. Coinjected ZIP potentiated theinhibitory action of PIR, whereas ZIP ${Delta}$ AID+ZZ and ZIP ${Delta}$ AID wereineffective. These results reinforce the central role playedby the PIR in Grb14 function.

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FIG. 10. Importance of Grb14-ZIP-PKC ${zeta}$ interaction in ZIP modulation of Grb14 action. Oocytes were coinjected with GST-Grb14 or GST-PIR in the presence of 100 ng of various GST-ZIP constructs, as indicated. Oocyte maturation in response to insulin was monitored as in Fig. 8. Data are the means + standard errors of the means (error bars) of three animals, each concerning 20 oocytes.

We have shown that phosphorylation of Grb14 increased its inhibitoryeffect on in vitro IR tyrosine kinase activity (Fig. 7). Wereasoned that if the role of ZIP is to allow Grb14 to be phosphorylatedby PKC ${zeta}$ , previous phosphorylation of the GST-PIR fusion wouldincrease its inhibitory effect on the action of insulin in theXenopus oocyte model. We thus tested the effect of decreasingamounts of GST-PIR, prephosphorylated or not, on insulin-inducedoocyte maturation. As shown in Fig. 11, the injection of 12.5ng of GST-PIR did not inhibit the effect of insulin. However,if the protein was prephosphorylated by recombinant PKC ${zeta}$ , theinjection of 12.5 ng, as well as 7.5 ng, totally inhibited theeffect of insulin. As a control, microinjection of the sameamounts of activated PKC ${zeta}$ in the absence of GST-PIR did not modifyinsulin-induced oocyte maturation. These results confirm invivo that the inhibitory action of Grb14 is increased when theprotein is phosphorylated.

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FIG. 11. PIR phosphorylation increases its inhibition of insulin-induced oocyte maturation. Oocytes were either microinjected with the indicated amounts of GST-PIR, which was (black bars) or was not (grey bars) previously phosphorylated by recombinant PKC ${zeta}$ as described in Materials and Methods, or microinjected with PKC ${zeta}$ alone (white bars). Oocyte maturation in response to insulin was monitored as in Fig. 8. Data are the means + standard errors of the means (error bars) of three animals, each concerning 20 oocytes.

All together, these data are consistent with the notion thatZIP acts as an adapter, linking PKC ${zeta}$ to Grb14 into a new signalingpathway.

DISCUSSION

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Discussion

The atypical protein kinase C isoforms (aPKCs), which includePKC ${zeta}$ and PKC ${lambda}$ , are implicated in multiple intracellular pathways.They regulate critical cellular functions such as cell proliferationand apoptosis (3, 12, 30, 49), and they are also important mediatorsof physiological actions of insulin such as translocation ofthe insulin-responsive glucose transporter GLUT4 and enhancementof glucose transport in adipocytes and skeletal muscle (1, 6,16, 65). Accumulating studies support the existence of adapterproteins coordinating physical association between PKC and substratesand thus acting as catalysts for specific reactions (47, 63).These proteins are key components for the determination of theselectivity of signaling events. Some of these effectors canmodulate aPKC catalytic activity, like lambda interacting proteinand Par-6 (partitioning-defective 6), acting as activators (10,56), or Par-4 (prostate androgen response-4), acting as an inhibitor(9). Atypical PKC isotype-specific interacting protein (ASIP)binds the aPKC catalytic domain (24). Interestingly, overexpressionof ASIP in 3T3-L1 adipocytes inhibits insulin-induced glucoseuptake (31). However, the mechanism by which ASIP inhibits PKC ${lambda}$ effect in GLUT4 translocation remains unclear. Another aPKC-interactingprotein, ZIP, binds selectively and constitutively to the V1portion of their regulatory domain and does not modulate theircatalytic activity (55, 60). ZIP contains a number of conservedmotifs, suggesting a role as a scaffold protein linking aPKCsto other proteins implicated in various signaling pathways.ZIP has been shown to recruit the proteins RIP and TRAF6, connectingPKC ${zeta}$ to the TNF- ${alpha}$ and IL-1 receptors, respectively (61, 62). Theseinteractions appear to be of functional relevance since theystimulate NF- ${kappa}$ B activity. In addition, it was recently reportedthat through its association with the NGF receptor TrkA, theZIP-TRAF6 complex is implicated in the NGF-induced NF- ${kappa}$ B activation(72). ZIP acts also as a link between PKC ${zeta}$ and the neuronal potassiumchannel Kvß2 (18).

In this study, we identified ZIP as a new downstream partnerof Grb14. Although we isolated two isoforms, ZIP ${alpha}$ and ZIPß,we did not find any functional difference between them. Theybound similarly to Grb14, and both of them potentiated the inhibitoryrole of Grb14 on insulin induced Xenopus oocyte maturation.Results of mapping studies indicated that the ZZ zinc fingerdomain of ZIP, which includes a novel atypical zinc finger motif(55), is required for the interaction with Grb14. This domainwas also recently shown to interact with the death domain kinaseRIP (62). All together, these results suggest that the ZZ zincfinger domain of ZIP is a new protein-protein interacting region.The PKC ${zeta}$ binding domain of ZIP, termed AID, is located N terminalof the ZZ zinc finger domain, indicating that ZIP can bind simultaneouslyGrb14 and PKC ${zeta}$ through two distinct but proximal modules. Usingcoimmunoprecipitation experiments in transfected CHO-IR andCOS cells, we confirmed that these interactions are not antagonistic,resulting in a ternary complex including Grb14, ZIP, and PKC ${zeta}$ .Importantly, Grb14, ZIP, and PKC ${zeta}$ form a heterotrimer also invivo in rat liver, demonstrating that endogenous proteins interactunder physiological conditions. By contrast with previous studiesreporting a ligand induction of ZIP multimeric complexes (61,62, 72), we were unable to detect any effect of insulin on theformation of this heterotrimer (Fig. 3A and data not shown).Nevertheless, the functional regulation by insulin of the Grb14-ZIP-PKC ${zeta}$ complex may involve other modifications, like phosphorylationsor conformational changes in response to the hormone. In thisregard, we reported that Grb14 undergoes insulin-stimulatedphosphorylation during labeling experiments in intact cells.Since we previously reported that Grb14 was not phosphorylatedon tyrosine residues under insulin stimulation in CHO-IR-Grb14cells (28), Grb14 is likely to be phosphorylated on serine/threonineresidues. This is in agreement with previous studies reportingthat Grb14 was phosphorylated in vivo mainly on serine residues(8, 58). In these studies, stimulation with PDGF (8) or fibroblastgrowth factor 2 (58) increased Grb14 phosphorylation to an extentsimilar to that after insulin stimulation. Other members ofthe Grb7 family of proteins are also phosphorylated on serine/threonineresidues (20). More particularly, it was shown that insulinincreased Grb10 serine phosphorylation, and it can be underlinedthat the effect of insulin on Grb10 and on Grb14 phosphorylationis of similar amplitude (13). The serine/threonine kinases involvedin Grb7 protein phosphorylation are still unidentified (20),but they do not appear to be either cyclic AMP- or calcium-stimulatedkinases (53) or classical or novel PKC isozymes (13, 53). Insulin-inducedGrb10 phosphorylation was blocked by wortmannin, suggestingthat insulin stimulates a kinase acting in a phosphatidyl inositol3-kinase dependent manner (13). Since insulin is known to stimulatePKC ${zeta}$ catalytic activity (5, 64, 65), and since ZIP anchors PKC ${zeta}$ and Grb14 into a heterotrimeric complex, we hypothesized thatGrb14 could be a substrate for the kinase. We thus tested whetherPKC ${zeta}$ could be implicated in insulin-stimulated Grb14 phosphorylation.In intact cells, insulin-stimulated Grb14 phosphorylation isdecreased by a PKC ${zeta}$ pseudosubstrate, which inhibits PKC ${zeta}$ enzymaticactivity. Furthermore, the overexpression of PKC ${zeta}$ increased insulin-inducedGrb14 phosphorylation. Together, these data suggest that theserine/threonine kinase PKC ${zeta}$ is a potential candidate for invivo insulin-induced Grb14 phosphorylation. However, a rolefor other kinases cannot be excluded, and further experimentsare needed to clarify this point. More particularly, the identityof kinases responsible for the high basal phosphorylation stateof these proteins is still unclear.

Of functional relevance, we provided evidence that phosphorylationof Grb14 increased its inhibitory effect on IR tyrosine kinaseactivity. Interestingly, among the various domains of Grb14,the PIR, which is responsible for both the specific interactionwith the activated IR tyrosine kinase loop and the inhibitionof the IR catalytic activity (2, 28), was preferentially phosphorylatedby PKC ${zeta}$ in vitro. Although this domain does not contain a conventionalphosphorylation motif for PKC, it exhibits 4 serine residuesfollowed in +1 by a hydrophobic amino acid, which has been shownto be present in all bona fide PKC phosphorylation sites (51).In addition, PKC ${zeta}$ was already described to phosphorylate IKKßin a nonconsensus motif (32). The identification of the Grb14-phosphorylatedsites and the mechanism whereby Grb14 phosphorylation increasesits inhibitory effect deserve further investigation. However,a simple hypothesis could be that this increased activity reflectsan increased binding affinity of the phosphorylated PIR forthe IR kinase loop.

Xenopus oocyte, which endogenously expresses insulin-signalingmolecules, provides a widely used experimental model to studyin vivo insulin action (15, 25, 59, 66). We show here that injectionof Grb14 into Xenopus oocyte specifically blocks insulin-inducedreinitiation of meiosis. These data correlate with Grb14 inhibitionof distal insulin effects, such as DNA or glycogen synthesis(28). By contrast, injection of ZIP alone did not alter oocytematuration. Since PKC ${zeta}$ is a critical step for the mitogenic signalin oocytes (3, 11), this result seems to confirm that ZIP doesnot regulate PKC ${zeta}$ activity in vivo. On the other hand, ZIP wasable to potentiate Grb14 inhibitory action. This effect requiredthe recruitment of PKC ${zeta}$ and could be mimicked by previous Grb14phosphorylation. These observations provide in vivo evidencethat ZIP acts as a functional scaffold protein connecting PKC ${zeta}$ to Grb14, allowing its phosphorylation. Interestingly, the PIRexpressed alone was sufficient to mediate both the inhibitoryeffect of Grb14 on insulin-induced reinitiation of meiosis andthe potentiation by ZIP of this inhibitory effect. This clearlydemonstrates that the interaction of the PIR with ZIP does notantagonize the regulation of its phosphorylation by PKC ${zeta}$ in vivo.These data also correlate with the observation that ZIP bindingto Grb14 did not alter the insulin-induced Grb14-IR interaction(data not shown). The PIR of Grb14 appears thus to be the functionalcore of the protein in the regulation of insulin signaling.Considering our in vitro and in vivo findings, a model emergeswhereby ZIP acts as a physical link in the assembly of a PKC ${zeta}$ -ZIP-Grb14complex, targeting the PKC ${zeta}$ activity to Grb14 (Fig. 12). Theconstitution of this complex has major functional consequences,since it potentiates the inhibitory effect of Grb14 on insulinsignaling.

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FIG. 12. A proposed model for the role of ZIP as an adapter connecting PKC ${zeta}$ to Grb14. ZIP interacts with the V1 domain of PKC ${zeta}$ through its AID domain, and with the PIR of Grb14 through its ZZ zinc finger domain, allowing the formation of a heterotrimeric complex. Under insulin stimulation, the PIR binds to the activated IR regulatory tyrosine kinase loop. PIR phosphorylation by PKC ${zeta}$ leads to an increased Grb14 inhibitory action on IR catalytic activity and insulin action.

PKC ${zeta}$ is known as a positive effector of the insulin signal transductionpathways, implicated in insulin-induced glucose transport, GLUT4translocation, and protein synthesis (1, 16, 43, 64, 65). However,recent studies reported that PKC ${zeta}$ can also play an inhibitoryrole in insulin signal transduction, by the inhibition of Akt(14, 39) or by the serine phosphorylation of IRS-1 (35, 57).Serine phosphorylation of IRS-1 by PKC ${zeta}$ participates in a negativefeedback control mechanism induced by insulin, which promotesthe dissociation of the IR-IRS-1 complex and leads to the terminationof insulin signal (35, 57). Interestingly, the present worksuggests that by enhancing the inhibitory activity of Grb14,PKC ${zeta}$ can use an additional way to down-regulate insulin signaling.

ACKNOWLEDGMENTS

We thank J. Camonis and R. Farese, respectively, for the kindgifts of the pFBL23 plasmid and the pcDNA3-PKC ${zeta}$ plasmid. We alsogratefully acknowledge V. Le Marcis for GST fusions productionand E. Clauser, T. Issad, and B. Desbuquois for critically readingthe manuscript.

This work was supported by Servier and by the Association pourla Recherche sur le Cancer (grant 5237 to A.-F. Burnol). B.C.is supported by a Servier fellowship, and V.B. is the recipientof a fellowship from the Ministère de la Recherche.

FOOTNOTES

* Corresponding author. Mailing address: Institut Cochin, CNRS-INSERM-Université René Descartes, Département d'Endocrinologie, 24 rue du Faubourg Saint-Jacques, 75674 Paris, France. Phone: 33 1 53 73 27 09. Fax: 33 1 53 73 27 03. E-mail: burnol{at}cochin.inserm.fr.

REFERENCES

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