Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14.

Grb14 belongs to the Grb7 family of adapters and was recently identified as a partner of the insulin receptor (IR). Here we show that Grb14 inhibits in vitro IR substrate phosphorylation. Grb14 does not alter the K(m) for ATP and behaves as an uncompetitive inhibitor for the IR substrate. Similar experiments performed with other members of the Grb7 family, Grb7 and Grb10, and with IGF-1 receptor argue in favor of a specific inhibition of the IR catalytic activity by Grb14. The IR-interacting domain of Grb14, the PIR, is sufficient for the inhibitory effect of Grb14, whereas the SH2 domain has no effect on IR catalytic activity. In Chinese hamster ovary (CHO) cells overexpressing both IR and Grb14, Grb14 binds to the IR as early as 1 min after insulin stimulation, and the two proteins remain associated. When interacting with Grb14, the IR is protected against tyrosine phosphatases action and therefore maintained under a phosphorylated state. However, the binding of Grb14 to the IR induces an early delay in the activation of Akt and ERK1/2 in CHO-IR cells, and ERK1/2 are less efficiently phosphorylated. These findings show that Grb14 is a direct inhibitor of the IR catalytic activity and could be considered as a modulator of insulin signaling.     

Lipid-protein interactions of growth factor receptor-bound protein 14 in insulin receptor signaling

Many retinal degenerative diseases show an early loss of rod cells followed by cone cells. In these degenerations the pathological phenotype is apoptosis. We have previously demonstrated the light-dependent tyrosine phosphorylation of the insulin receptor in the retina, which leads to the activation of anti-apoptotic signaling molecules. The mechanism of the regulation of the insulin receptor in the retina is not known. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of the retinal insulin receptor (IRbeta) identified a member of the Grb7 (growth factor receptor-bound protein 7) gene family, Grb14. In this report, we describe the unique features of Grb14. Grb14 forms a specific complex with the cytoplasmic domain of IRbeta when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase activity. Deletion mutagenesis on Grb14 indicated a phosphorylated insulin receptor interacting (PIR) domain between the PH (pleckstrin homology) and SH2 (Src homology) domains that binds to IRbeta. Nuclear import assays in yeast indicated the presence of a functional nuclear localization signal in Grb14 between amino acids 63 and 68 (RRKKD). Subcellular localization of isolated retinas probed with anti-Grb14 antibody further confirmed the presence of Grb14 in nuclear fractions. Analysis using a protein-lipid overlay assay indicated binding of Grb14 and its PH domain to D3 phosphoinositides. In addition, Grb14-phosphoinositide 3,4,5-trisphosphate complexes are detected in lysates prepared from insulin-stimulated retina tissues, whereas Grb14-phosphoinositide 4,5-bisphosphate interactions are observed under non-insulin stimulated conditions. These findings suggest that Grb14 could be a diverse regulator of insulin receptor mediated pathways in the retina.     

Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.     

Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14(-/-) studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling in a tissue-specific manner. In this study, we made a novel finding that Grb14 inhibits the activity of PTP1B, the major negative regulator of insulin receptor (IR) signaling, in a phosphorylation-regulated manner. Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for interaction with PTP1B, resulting in the competitive inhibition of PTP1B activity. We also found that rhodopsin-regulated Src kinase activation in retina leads to the phosphorylation of Grb14. Further, ablation of Grb14 resulted in significantly elevated retinal PTP1B activity in vivo. PTP1B is known to be regulated by oxidation, glutathionylation, phosphorylation, and SUMOlyation, and our study for the first time demonstrates the inhibition of PTP1B activity in vivo by protein molecule Grb14 in a tissue-specific manner.     

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.     

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains. The isolated PIR and PIR-SH2 domains were physiologically active and inhibited insulin-induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on (15)N-labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins.     

SAXS study of the PIR domain from the Grb14 molecular adaptor: a natively unfolded protein with a transient structure primer?

Grb14 belongs to the Grb7 family of adapters and was identified as a negative regulator of insulin signal transduction. Between the PH (pleckstrin homology) and SH2 (Src homology 2) domains is a new binding domain implicated in the interaction with receptor tyrosine kinases called PIR (phosphorylated insulin receptor interaction region). Both PIR and SH2 domains interact with the insulin receptor, but their relative role varies considering the member of the Grb7 family and the tyrosine kinase receptor. In the case of Grb14, PIR is the main binding domain and is sufficient to inhibit the insulin receptor kinase activity. We have proposed, on the basis of NMR measurements, that PIR lacks ordered structure and presents a high flexibility, although remaining fully active. To complement this first study, we have used small-angle x-ray scattering in solution together with a modeling approach representing the PIR domain as a chain of pseudo residues. Circular dichroism experiments were also performed in the presence of variable amounts of trifluoroethanol. These observations, together with an ensemble of sequence analyses and previous NMR results, all support the view of PIR as essentially unstructured but with a potentially structured short stretch encompassing residues 399-407. This stretch, which may be only structured transiently in the isolated molecule, could play a major role in Grb14 PIR binding to a biological partner by undergoing a structural transition.     

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.     

Inhibition of FGF receptor signalling in Xenopus oocytes: differential effect of Grb7, Grb10 and Grb14

The role of Grb7 adapters, Grb7, Grb10, and Grb14, was investigated in Xenopus oocytes expressing fibroblast growth factor receptors (FGFR). FGF-induced maturation of FGFR-expressing oocytes was blocked by previous injection of Grb7 or Grb14, but not Grb10. This effect correlated with Grb7/14 binding to the receptor, and inhibition of the Ras-dependent pathway. Interestingly, the phosphorylated insulin receptor interacting region (PIR) and Src 2 homology domains (SH2) of Grb7 and Grb14 were differently implicated in the inhibition of FGFR signalling. This study provided further evidence for specificity of the biological action of the Grb7 adapters on receptor tyrosine kinase signalling.     

Characterization of PDK2 activity against protein kinase B gamma

Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBgamma identified protein kinase C (PKC) zeta, an atypical PKC, as an interactor with PKBgamma, an association requiring the pleckstrin homology domain of PKBgamma. Endogenous PKBgamma was shown to associate with endogenous PKCzeta both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCzeta, whether endogenous PKCzeta from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCzeta from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBgamma, in vitro. In vivo, overexpression of PKCzeta stimulated the phosphorylation of approximately 50% of the PKBgamma molecules, suggesting a physiologically meaningful effect. However, pure PKCzeta protein was incapable of phosphorylating S472 of PKBgamma. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCzeta immunoprecipitates. Staurosporine inhibited the PKCzeta activity but not the PDK2 activity in PKCzeta immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCzeta and that PKCzeta, by binding PKBgamma, functions to deliver the PDK2 to a required location. PKCzeta thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBgamma. Because both PKCzeta and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCzeta, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.     

Identification of a NPXY motif in growth factor receptor-bound protein 14 (Grb14) and its interaction with the phosphotyrosine-binding (PTB) domain of IRS-1

Recently we have shown that insulin fails to induce the phosphorylation of IRS-1 in the retina [Rajala et al. (2004) Biochemistry 43, 5637-5650], even though there is widespread expression of IRS-1 throughout the retina. These results suggest the expression of tissue-specific regulators in the retina. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal insulin receptor identified a novel member of the Grb7 gene family, Grb14. Phosphorylation prediction software indicated 6 out of 18 tyrosine residues were most likely to be phosphorylated. Out of six tyrosine phosphorylation sites, one of the tyrosine residues in Grb14 is present in a conserved sequence motif, FXNPXY. The NPXY motifs are recognized by proteins containing a domain known as phosphotyrosine-binding (PTB) or phosphotyrosine-interacting domain (PID). The biological function of the PTB domain is to drive recruitment of signaling adapters such as IRS-1 or Shc to NPXpY (pY stands for phosphotyrosine) on activated receptor tyrosine kinases. We have made a novel finding that the PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. In addition, Grb14-IRS-1 complexes are detected in lysates prepared from retina tissues. We suggest that the Grb14 NPXY motif could be acting as a dominant negative for IRS-1 functions in the retina, and this hypothesis is consistent with the recent study that Grb14-deficient mice exhibit enhanced IRS-1 phosphorylation and activation of protein kinase B. This is the first report describing the presence of the NPXY motif in Grb14 and binding of the PTB domain of IRS-1 in a phosphorylation-independent manner.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Background

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?

Results

Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2^Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.

Conclusions

Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.

     

Interaction with Grb14 results in site-specific regulation of tyrosine phosphorylation of the insulin receptor

The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.     

Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.     

Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10

Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.     

Evaluation of the role of protein kinase Czeta in insulin-induced heart 6-phosphofructo-2-kinase activation

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK) that phosphorylates and activates heart 6-phosphofructo-2-kinase (PFK-2) was purified from serum-fed HeLa cells and found to contain protein kinase Czeta (PKCzeta). Both WISK and recombinant PKCzeta were inhibited by a pseudo-substrate peptide inhibitor of PKCzeta. WISK and PKCzeta phosphorylated and activated recombinant heart PFK-2 by increasing its Vmax. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCzeta phosphorylated only Thr475. In perfused rat hearts, insulin activated protein kinase B (PKB) 16-fold compared with the untreated controls. However in the same experiments, no change in phosphorylation state of the activation loop Thr410 residue of PKCzeta was observed. By contrast, in incubations of isolated rat epididymal adipocytes, where insulin activated PKB 30-fold compared with the untreated controls, a 50% increase in PKCzeta Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCzeta construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCzeta is required for PFK-2 activation by insulin in heart.     

Mechanism of SHIP-mediated inhibition of insulin- and platelet-derived growth factor-stimulated mitogen-activated protein kinase activity in 3T3-L1 adipocytes

The Src homology 2-containing 5' inositolphosphatases (SHIP and SHIP2) dephosphorylate 3'-phosphorylated PtdIns on the 5' position, decreasing intracellular levels of PtdIns 3,4,5-P3. In the current study, we investigated the role of SHIP in insulin and platelet-derived growth factor (PDGF) signaling by expressing wild-type (WT) and catalytically inactive SHIPDeltaIP in 3T3-L1 adipocytes, utilizing adenoviral infection. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIPDeltaIP, and tyrosine phosphorylation of SHIP-associated proteins increased after ligand stimulation. Tyrosine-phosphorylated PDGFR, IR, and insulin receptor substrate-1 all immunoprecipitated with SHIP. Expression of WT and DeltaIP mutant SHIP did not affect tyrosine phosphorylation of either the insulin or the PDGF receptor, or the expression of insulin receptor substrate-1 and Shc proteins. Both SHIP-WT and SHIPDeltaIP blocked insulin and PDGF-induced MAPK and MAPK kinase phosphorylation as well as, GTP-bound Ras activity, suggesting that the catalytic activity of SHIP is not necessary for these effects. SHIP associated with Shc upon ligand stimulation, indicating that the SHIP-Shc association is phosphorylation dependent. This association was primarily between the SHIP-SH2 domain and the phosphorylated tyrosine residues of Shc because no association was observed when the 3YF-Shc mutant was coexpressed with SHIP. The Shc*Grb2 association was not compromised by SHIP expression, despite complete inhibition of the Ras/MAPK pathway. Interestingly, son-of-sevenless (SOS) protein normally found in Grb2 complexes was markedly reduced in SHIP expressing cells, whereas the displaced SOS was recovered when the post-Grb2-IP supernatants were blotted with anti-SOS antibody. Thus, SHIP competes son-of-sevenless (SOS) away from Shc-Grb2. In summary, 1) SHIP-WT and SHIPDeltaIP expression inhibit insulin and PDGF stimulated Ras, MAPK kinase, and MAPK activities; 2) SHIP associates with tyrosine phosphorylated Shc, and the proline-rich sequences in SHIP associate with Grb2 and titrate out SOS to form Shc*Grb2*SHIP complexes; and 3) dissociation of SOS from the Shc*Grb2 complex inhibits Ras GTP loading, leading to decreased signaling through the MAPK pathway.     

Structural basis for inhibition of the insulin receptor by the adaptor protein Grb14

Grb14, a member of the Grb7 adaptor protein family, possesses a pleckstrin homology (PH) domain, a C-terminal Src homology-2 (SH2) domain, and an intervening stretch of approximately 45 residues known as the BPS region, which is unique to this adaptor family. Previous studies have demonstrated that Grb14 is a tissue-specific negative regulator of insulin receptor signaling and that inhibition is mediated by the BPS region. We have determined the crystal structure of the Grb14 BPS region in complex with the tyrosine kinase domain of the insulin receptor. The structure reveals that the N-terminal portion of the BPS region binds as a pseudosubstrate inhibitor in the substrate peptide binding groove of the kinase. Together with the crystal structure of the SH2 domain, we present a model for the interaction of Grb14 with the insulin receptor, which indicates how Grb14 functions as a selective protein inhibitor of insulin signaling.     

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.