Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.     

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.     

Protein kinase A-mediated 14-3-3 association impedes human Dapper1 to promote dishevelled degradation

Wnt signaling regulates embryo development and tissue homeostasis, and its deregulation leads to an array of diseases, including cancer. Dapper1 has been shown to be a key negative regulator of Wnt signaling. However, its function and regulation remain poorly understood. In this study, we report that 14-3-3β interacts with human Dapper1 (hDpr1). The interaction is dependent on protein kinase A (PKA)-mediated phosphorylation of hDpr1 at Ser-237 and Ser-827. 14-3-3β binding attenuates the ability of hDpr1 to promote Dishevelled (Dvl) degradation, thus enhancing Wnt signaling. We further provide evidence that PKA-mediated Dpr1 phosphorylation may contribute to growth and tumor formation of colon cancer Caco2 cells. Finally, we show that cyclooxygenase-2 expression and PKA activation are positively correlated with Dvl protein levels in colon cancer samples. Together, our findings establish a novel layer of regulation of Wnt signaling by PKA via the 14-3-3-Dpr1-Dvl axis.     

Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance. However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine. One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1. During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1. In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades. These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.     

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation

Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.     

Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle

Certain PKC isoforms are stimulated by insulin and interact with IR as well as with IRS, but it is still not clear if specific PKC isoforms regulate IR signaling directly or through IRS-1. PKCalpha may regulate IRS activity in response to insulin. We investigated the possibility that PKCalpha may be important in insulin signaling. Studies were conducted on skeletal muscle in adult mice and on L6 skeletal cells. PKCalpha is constitutively associated with IRS-1, and insulin stimulation of PKCalpha causes disassociation of the two proteins within 5 min. Blockade of PKCalpha inhibited insulin-induced disassociation of PKCalpha from IRS1. Selective inhibition of PKCalpha increased the ability of insulin to reduce blood glucose levels. Insulin stimulation activates PKB and increases the association of PKCalpha with PKB. Blockade of PKCalpha increased threonine phosphorylation of PKB. We suggest that PKCalpha regulates insulin signaling in skeletal muscle through its disassociation from IRS-1 and association with PKB.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.     

Protein kinase C-zeta-induced phosphorylation of Ser318 in insulin receptor substrate-1 (IRS-1) attenuates the interaction with the insulin receptor and the tyrosine phosphorylation of IRS-1

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.     

14-3-3 binding regulates catalytic activity of human Wee1 kinase.

The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15. Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15. Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions. Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function. We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01. A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo. These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.     

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

Impairment of bone healing by insulin receptor substrate-1 deficiency

Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1(-/-) mice). Among 15 IRS-1(-/-) mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1(-/-) fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1(-/-) chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1(-/-) culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.     

14-3-3 facilitates insulin-stimulated intracellular trafficking of insulin receptor substrate 1

The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.