3-Phosphohistidine cannot replace phosphotyrosine in high-affinity binding to phosphotyrosine binding or Src homology 2 domains

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.     

The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1

Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions were not required for these insulin actions; however, the IH2(PTB) improved the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis. The sensitivity of these responses was partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.     

The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin-1 and dynamin-2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin-1 and dynamin-2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.     

A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain

A novel Xenopus insulin receptor substrate cDNA was isolated by hybridization screening using the rat insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (PH) domain and phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u contains several potential Src homology 2 (SH2)-binding sites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with the previously identified xIRS-1 and the four members of the mammalian IRS family (1 through 4) indicated that xIRS-u has similar overall sequence homology (33-45% identity) to all mammalian IRS proteins. In contrast, the previously isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to the other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence analyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA encoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expression of a 120-kDa protein (including 5 copies of the 13-amino acid Myc tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP kinase activation with a concomitant acceleration of insulin-induced oocyte maturation. An aminoterminal deletion of the PH domain (xIRS-u deltaPH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.     

The insulin receptor substrate 1 associates with phosphotyrosine phosphatase SHPTP2 in liver and muscle of rats

OBJECTIVE: To determine efficacy of a vaccine containing modified-live bovine viral diarrhea virus (BVDV) type 1 for protecting pregnant cows and their fetuses against virulent heterologous BVDV type 1. DESIGN: Randomized controlled cohort study. ANIMALS: 18 yearling beef heifers seronegative for BVDV and negative when tested for BVDV by virus isolation. PROCEDURE: Cattle were randomly assigned to control (unvaccinated; n = 6) or vaccinated (12) groups. Vaccinated heifers were given a combination vaccine containing modified-live BVDV type 1 comprising a cytopathic (NADL) strain. All 18 heifers were then bred and challenge-exposed between 70 and 75 days of gestation with BVDV type 1, administered intranasally. Cattle were monitored, and infection status of offspring was determined after parturition. Antibody concentrations of vaccinated and control heifers were also monitored. RESULTS: All 6 calves from control heifers had positive results on multiple virus isolation tests and were considered persistently infected. In comparison, only 2 calves from vaccinated cows had positive results on virus isolation tests and were considered persistently infected. One vaccinated heifer aborted, but the fetus was not persistently infected, and the abortion was not attributed to BVDV infection. CLINICAL IMPLICATIONS: Analysis of these data indicated that a single dose of a modified-live NADL-derived BVDV type 1 vaccine will confer protection to dams and their fetuses against challenge-exposure to heterologous BVDV type 1 organisms.     

Binding of G protein beta gamma-subunits to pleckstrin homology domains

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.     

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein

Psychological assessments made at the time of bone marrow transplantation (BMT) were examined in 31 patients at 1-2 years posttransplantation and factors associated with survival were explored. Psychological assessments were carried out soon after admission to hospital for the BMT and about 3-4 weeks later. Cox regression survival analysis revealed that an interactive biopsychological model could explain survival status. Shorter survival was associated with mismatched marrow grafts (p = 0.04), prior experience with chemoradiotherapy (p-0.02), disease stage (p = 0.04), higher symptom distress during BMT (p = 0.008), less hopefulness (p = 0.005), and more acceptance of the situation (p = 0.02). These psychological/psychosomatic and personality characteristics may act directly by interacting with immune function or indirectly by leading to other behaviors known to affect survival in cancer patients. Enhancing more effective coping strategies and altering high symptom-related distress during BMT seem important means not only for improving psychosocial adjustment (quality of life), as has been shown in the literature, but also for increasing survival (quantity of life).     

Dual specificity of Src homology 2 domains for phosphotyrosine peptide ligands

SH2 domains mediate protein-protein interactions and are involved in a wide range of intracellular signaling events. SH2 domains are 100-amino acid stretches of protein that bind to other proteins containing phosphotyrosine residues. A current major research goal is formulation of the structural principles which govern peptide-binding specificity in SH2 domains. Several structures (both X-ray and NMR) of SH2 domains have now been determined. Short peptide fragments on the carboxyl-terminal side of the phosphotyrosine residue carry the sequence specific information for SH2 recognition. The bound peptides are held in an extended conformation. However, for the GRB2 SH2 domain, the peptide adopts a beta-turn as the motif for recognition [Rahuel, J., et al. (1996) Nat. Struct. Biol. 3, 586-589]. Our SAR data and molecular modeling studies suggest that many SH2 domains, such as the SH2 domains of Lck, Src, and p85, can interact with high affinity with short peptide sequences at least in two ways which are sequence-dependent. The peptide forms either an extended chain across the D-strand of SH2 domains with anchors at pY and pY+3 or, as in the case of GRB2 SH2, a beta-turn with anchors at pY and pY+2. Due to a bulky tryptophan in its EF1 loop, GRB2 SH2 cannot bind peptide conformations such as the extended chain and thus has a unique specificity.     

Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition

The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T2 protein were constructed. One mutant, deltaL113, containing intact copies of only two cysteine-rich domains, was not secreted and was incapable of binding rabbit TNF-alpha yet retained full ability to inhibit virus-induced apoptosis of RL-5 cells. Thus, the minimal domain of intracellular M-T2 protein required to inhibit apoptosis is distinct from that required by the extracellular M-T2 for functional TNF-alpha binding and inhibition. This is the first report of a virus-encoded immunomodular protein with two distinct antiimmune properties.     

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an "unregulated" mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.     

Inhibition of mSOS-activity by binding of phosphatidylinositol 4,5-P2 to the mSOS pleckstrin homology domain

There are several recently reported examples of inositol phospholipids binding to pleckstrin homology (PH) domains of proteins. The PH domain of SOS, a guanine nucleotide exchange factor for Ras, binds to phosphatidylinositol 4,5 bisphosphate (PtdIns4,5P2). We found that binding of PtdIns4,5P2 to 6-his-tagged recombinant mSOS in vitro inhibits the ability of SOS to catalyze the association of GTP on p21RAS. This inhibition was specific for PtdIns4,5P2: a number of other phosphatidylinositols and phosphatidylserine failed to inhibit Ras GTP-association. We confirmed that the specificity of binding of PtdIns's to recombinant GST-SOS-PH domain is the same as the specificity of PtdIns's for inhibition of SOS activity: namely, that only PtdIns4,5P2 binds significantly to the SOS-PH domain. In addition, the inhibition of Ras GTP-binding is not blocked by excess free inositols suggesting that SOS binds to PtdIns4,5P2 with higher affinity than it binds to free inositols. Addition of SOS-PH domain protein prevented the inhibition of SOS by PtdIns4,5P2 as did addition of the high affinity PtdIns4,5P2-binding drug neomycin. This confirmed that SOS inhibition is mediated by the SOS-PH domain binding to the inositol moiety of PtdIns4,5P2. Binding of Grb2 to SOS did not prevent the inhibition of SOS by PtdIns4,5P2 suggesting that there must be another mechanism for regulating this inhibition. These findings show that the phospholipid PtdIns4,5P2 can suppress the activity of an enzyme involved in signal transduction and suggest that this inhibitory effect must be relieved when SOS is activated.     

Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.     

Ethanol inhibits hepatocyte proliferation in insulin receptor substrate 1 transgenic mice

BACKGROUND & AIMS: Long-term ethanol consumption is known to impair the ability of the liver to regenerate, but the molecular mechanisms are poorly understood. Multiple growth factors promote hepatocyte proliferation, some of which involve the insulin receptor substrate 1 (IRS-1)-mediated signal transduction pathway. To explore effects of ethanol on the IRS-1 signal liver growth in vivo, studies in transgenic mice overexpressing IRS-1 in the liver were performed because these mice show constitutive activation of the downstream signal transduction pathways leading to enhanced hepatocyte proliferation. METHODS: Tyrosyl phosphorylation of IRS-1 and subsequent protein-protein interactions were examined in liver lysates from animals fed ethanol or control diet. Activity of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) was assessed by specific enzymatic assays. Hepatocyte proliferation was measured by incorporation of [3H]thymidine into liver DNA. RESULTS: Tyrosyl phosphorylation of IRS-1, association of IRS-1 with PI3K, and activation of downstream PI3K and MAPK pathways were greatly reduced as a result of long-term ethanol consumption. Ethanol virtually abolished the enhanced hepatocyte DNA synthesis induced by expression of the IRS-1 transgene. CONCLUSIONS: Altered transmission of growth signals through the IRS-1-mediated signal transduction cascade may represent a molecular mechanism of how ethanol inhibits hepatocyte proliferation.     

IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance in obesity and diabetes through its ability to decrease the tyrosine kinase activity of the insulin receptor (IR). Treatment of cultured murine adipocytes with TNF-alpha was shown to induce serine phosphorylation of insulin receptor substrate 1 (IRS-1) and convert IRS-1 into an inhibitor of the IR tyrosine kinase activity in vitro. Myeloid 32D cells, which lack endogenous IRS-1, were resistant to TNF-alpha-mediated inhibition of IR signaling, whereas transfected 32D cells that express IRS-1 were very sensitive to this effect of TNF-alpha. An inhibitory form of IRS-1 was observed in muscle and fat tissues from obese rats. These results indicate that TNF-alpha induces insulin resistance through an unexpected action of IRS-1 to attenuate insulin receptor signaling.     

Differential association of the pleckstrin homology domains of phospholipases C-beta 1, C-beta 2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins

Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-delta and PLC-gamma have been studied, the comparable domains of the beta isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-beta 1 and -beta 2 (PH-beta 1 and PH-beta 2, respectively) for lipid bilayers and G-beta gamma subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-beta gamma subunits had little affect on their membrane affinity. In contrast, binding of PLC-delta 1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-beta gamma subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-beta gamma were in the following order: PH-beta 2 >/= PH-beta 1 > PH-delta 1; the affinities of the native enzyme were as follows: PLC-beta 2 > PLC-delta 1 > PLC-beta 1. Thus, the PH domain of PLC-beta 1 interacts with G-beta gamma in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-beta2 with G-beta gamma is comparable to that of the full-length protein and may play a key role in G-beta gamma recognition.     

Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics

Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of phosphotyrosine mimetics, the (difluoronaphthylmethyl)phosphonic acids and the fluoromalonyl tyrosines, have been determined to resolutions greater than 2.3 A. The fluoromalonyl tyrosine residue was incorporated within a cyclic hexapeptide modeled on an autophosphorylation site of the epidermal growth factor receptor. The structure of this inhibitor bound to PTP1B represents the first crystal structure of a non-phosphonate-containing inhibitor and reveals the mechanism of phosphotyrosine mimicry by the fluoromalonyl tyrosine residue and the nature of its interactions within the catalytic site of PTP1B. In contrast to complexes of PTP1B with phosphotyrosine-containing peptides, binding of the fluoromalonyl tyrosine residue to the catalytic site of PTP1B is not accompanied by closure of the catalytic site WPD loop. Structures of PTP1B in complex with the (difluoronaphthylmethyl)phosphonic acid derivatives reveal that substitutions of the naphthalene ring modulate the mode of inhibitor binding to the catalytic site and provide the potential for enhanced inhibitor affinity and the generation of PTP-specific inhibitors. These results provide a framework for the rational design of higher affinity and more specific phosphotyrosine mimetic inhibitors of not only protein tyrosine phosphatases but also SH2 and PTB domains.     

The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes

BACKGROUND: Peripheral membrane proteins are targeted to the cytoplasmic face of specific intracellular membranes. The organelle-specific ligands recognised by peripheral proteins include other proteins and lipids. Oxysterol-binding protein (OSBP) translocates from the cytoplasm to the Golgi apparatus on binding oxygenated derivatives of cholesterol. The mechanism by which OSBP recognises the Golgi is unknown. It does, however, contain a pleckstrin homology (PH) domain, which in other proteins has been found to mediate regulated membrane binding, although in all previously studied examples the binding is to the plasma membrane. RESULTS: The PH domain of OSBP and of a yeast homologue, Osh1p, were sufficient to target proteins specifically to mammalian Golgi membranes. In addition, high level expression disrupted Golgi architecture and prevented forward traffic of cargo protein. In vitro, the OSBP PH domain bound to Golgi membranes in a manner apparently dependent on phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or a related phosphatidylinositide. The OSBP PH domain bound to PI(4,5)P2 in liposomes with a submicromolar dissociation constant. CONCLUSIONS: The PH domains of OSBP and its yeast homologue recognise a determinant which is specific to Golgi membranes and important for Golgi function. The determinant appears to be a combination or a phosphatidylinositol polyphosphate and a second, Golgi-specific feature.     

Mechanism of insulin action--signal transductions after binding to insulin receptor

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.     

The role of the Src homology domains in morphological transformation by v-src

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.