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Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.  相似文献   

3.
PHIP是一种与胰腺β细胞中胰岛素受体底物(IRS)的PH结构域相互作用的蛋白。根据小鼠PHIP(mPHIP)mRNA翻译的不同起始位点,除全长的PHIP1外,mPHIP基因还编码其他3种不同变异体。在胰岛素诱导的信号途径中,主要分布于细胞核的PHIP1和IRS-1的PH结构域相互作用,介导IRS蛋白酪氨酸的磷酸化。IRS-2和PHIP1的共表达能诱导IRS在细胞膜上的定位,促进葡萄糖转运蛋白4(GLUT4)向细胞质膜的转移。PHIP1的表达能提高β-细胞内细胞周期蛋白D2的表达,促进β细胞的生长。PHIP1的表达活化蛋白激酶B(PKB),活化的PKB能明显抑制β细胞的凋亡。PHIP与胰岛素信号传导途径中其他信号分子的相互作用机制尚不明确。  相似文献   

4.
Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.  相似文献   

5.
The pleckstrin homology (PH) domain-containing protein casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. How CKIP-1 localization is determined remains largely unclear. We observed that isolated CKIP-1-PH domain was predominantly localized in the nucleus and the C-terminus of CKIP-1 counteracted its nuclear localization. The net charge of basic residues and a serine-rich motif within the PH domain plays a pivotal role in the localization switch of both full-length CKIP-1 and the isolated PH domain. We propose that the N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its subcellular localization and the nucleus-plasma membrane shuttling.  相似文献   

6.
Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.  相似文献   

7.
Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.  相似文献   

8.
Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal, which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to D-myo-phosphatidylinositol 3,4,5-trisphosphate or D-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.  相似文献   

9.
To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.  相似文献   

10.
Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ~100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.  相似文献   

11.
In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.  相似文献   

12.
One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-γ (PLC-γ) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.  相似文献   

13.
The amino terminus of phospholipase D1 (PLD1) contains three potential membrane-interacting determinants: a phox homology (PX) domain, a pleckstrin homology (PH) domain and two adjacent cysteines at positions 240 and 241 within the PH domain that are fatty acylated in vivo. To understand how these determinants contribute to membrane localization, we have mutagenized critical residues of the PLD1 PH domain in the wild type or palmitate-free background in the intact protein, in a fragment that deletes the first 210 amino acids including the PX domain, and in the isolated PH domain. Mutants were expressed in COS-7 cells and examined for membrane residence, intracellular localization, palmitoylation, and catalytic activity. Our results are as follows. 1) Mutagenesis of critical residues of the PH domain results in redistribution of PLD1 from membranes to cytosol, independently of fatty acylation sites. Importantly, PH domain mutants in the wild type background showed greatly reduced fatty acylation, despite the presence of all relevant cysteines. 2) The isolated PH domain did not co-localize with PLD1 and was not palmitoylated. 3) The PX deletion mutant showed similar distribution and palmitoylation to the intact protein. Interestingly, PH domain mutants in this background showed significant palmitoylation and incomplete cytosolic redistribution. 4) PH domain mutants in the wild type or palmitate-free background maintained catalytic activity. We propose that membrane targeting of PLD1 involves a hierarchy of signals with a functional PH domain allowing fatty acylation leading to strong membrane binding. The PX domain may modulate function of the PH domain.  相似文献   

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The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.  相似文献   

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In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.  相似文献   

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In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms.  相似文献   

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