Chromosomal and genomic organization of Ty1-copia-like retrotransposon sequences in the genus Avena

Regulation of ligand-mediated signal transduction through transmembrane tyrosine kinase growth factor receptors involves phosphorylation of tyrosine residues in the intracellular domain of the receptor. The insulin-like growth factor-I (IGF-I) receptor contains three tyrosine residues in the carboxy-terminal domain at positions 1250, 1251, and 1316. Of these, only the tyrosine at position 1316 is conserved in the homologous position of the insulin receptor. Mutational analysis was used to study the role of these tyrosines in specific outcomes of IGF-I-mediated signal transduction. Mutations in the human IGF-I receptor were either replacement of tyrosines 1250 and 1251 with phenylalanine and histidine (yyFH), respectively, or replacement of the conserved distal tyrosine (position 1316) with phenylalanine (yCF). The yyFH mutation results in an IGF-I receptor with the amino acids found in the homologous position of the human insulin receptor. Cells overexpressing mutated IGF-I receptors were compared with cells expressing only endogenous IGF-I receptors or overexpressing wild-type IGF-I receptors. The ability of yyFH mutant IGF-I receptors to autophosphorylate the beta-subunit or phosphorylate insulin receptor substrate-1 was not significantly different from wild-type type IGF-I receptors. However, one or both of the proximal tyrosine residues (positions 1250 and 1251) in the carboxy-terminus of the IGF-I receptor are essential for IGF-I-stimulation of mitogenic and tumorigenic pathways. IGF-I-induced mitogenesis, measured as thymidine incorporation and cellular proliferation, was abrogated in cells overexpressing mutant IGF-I receptors with replacement of the proximal double tyrosines (positions 1250 and 1251). Fibroblasts expressing this mutant IGF-I receptor formed fewer tumors than the negative control cells, whereas cells expressing wild-type IGF-I receptors formed large tumors in all recipient mice injected. Conversely, cells expressing mutant IGF-I receptors with only the conserved distal tyrosine (position 1316) replaced had slightly reduced IGF-I-stimulated beta-subunit autophosphorylation, thymidine incorporation, and cellular proliferation when compared with cells expressing wild-type receptors. Phosphorylation of insulin receptor substrate-1 by the yCF mutant receptors was not impaired. Despite the ability of these mutant receptors to stimulate mitogenic growth, fibroblasts expressing this mutant receptor were also incapable of forming tumors in recipient nude mice. The distal tyrosine (position 1316) of the IGF-I receptor is crucial for tumor formation but is not essential for IGF-I stimulated mitogenesis. Thus, the tyrosine moieties in the carboxy-terminus of the IGF-I receptor participate in the signal transduction pathways that affect the mitogenic and tumorigenic potentials of cells expressing mutant IGF-I receptors.     

Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells

Nutrients and certain growth factors stimulate pancreatic beta-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic beta-cell line, INS-1, it was found that glucose (6-18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6-18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose + 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3'-kinase (PI 3'-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3'-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced beta-cell proliferation. In contrast, p70(S6K) was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced beta-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3'-kinase activity and downstream activation of p70(S6K). The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes beta-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.     

Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization

In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Insulin receptor substrate 2 and Shc play different roles in insulin-like growth factor I signaling

The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3'-kinase complex may regulate growth cone extension and membrane ruffling.     

Requirement for phosphatidylinositol 3'-kinase activity in platelet-derived growth factor-stimulated tyrosine phosphorylation of p125 focal adhesion kinase and paxillin

The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF-stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr-740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3'-kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

Blocking ligand occupancy of the alphaVbeta3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells

Blocking alphaVbeta3 integrin occupancy results in attenuation of the cellular migration response to insulin-like growth factor I (IGF-I). To determine whether integrin antagonists alter other IGF-I-stimulated biologic actions, quiescent smooth muscle cells (SMCs) were exposed to echistatin and their ability to respond to IGF-I was determined. Echistatin (10(-7) M) inhibited IGF-I-stimulated DNA synthesis by 80%, and the protein synthesis response also was inhibited. Therefore blocking occupancy of alphaVbeta3 inhibited multiple target cell actions of IGF-I. To determine whether blocking alphaVbeta3 occupancy could alter IGF-I receptor-mediated signal transduction, the ability of IGF-I to stimulate phosphorylation of insulin receptor substrate-1 (IRS-1) was analyzed. A 10-min exposure to 100 ng/ml of IGF-I resulted in a substantial increase in phosphorylated IRS-1, and echistatin (10(-7) M) blocked the IGF-I-induced IRS-1 phosphorylation response. Echistatin also attenuated downstream signaling because the capacity of the p85 subunit of phosphatidylinositol-3 kinase (PI-3 kinase) to bind to IRS-1 was blocked. In contrast, exposure of SMCs to vitronectin (1.0 micrograms/cm2) or thrombospondin (0.25 micrograms/cm2), two known ligands for alphaVbeta3, resulted in enhancement of the IGF-I-stimulated IRS-1 response. To determine whether these effects were caused by alterations in receptor kinase activity, the IGF-I receptor was immunoprecipitated and then analyzed for phosphotyrosine. Echistatin (10(-7) M) significantly reduced IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor beta subunit. We conclude that occupancy of the alphaVbeta3 integrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor and phosphorylate IRS-1. Activation of the alphaVbeta3 receptor results in an interaction with the IGF-I signal transduction pathway, which modulates SMCs responsiveness to IGF-I.     

Characterization of the novel focal adhesion kinase RAFTK in hematopoietic cells

Protein tyrosine kinases (PTKs) mediate signals that respond to many pivotal cellular functions. Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and PTKs, is a critical control mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion kinase (FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signaling and plays a role in signal transduction pathways mediating cell adhesion, motility and anchorage-independent growth. Recently, we and others have identified a novel protein tyrosine kinase termed RAFTK, (also known as Pyk2 or Cak-beta), which is related to FAK. This review describes the role of RAFTK in various signaling cascades mainly in reference to hematopoietic cell lineages.     

The phosphotyrosine phosphatase SHP-2 participates in a multimeric signaling complex and regulates T cell receptor (TCR) coupling to the Ras/mitogen-activated protein kinase (MAPK) pathway in Jurkat T cells

Src homology 2 (SH2) domain-containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase-mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-zeta chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2-associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3'-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3'-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.     

Adhesion molecules during immune response to exercise

PURPOSE: The extracellular matrix serves as a structural support for the corneal stroma and mediates signaling events that regulate the intracellular environment of stromal keratocytes. We hypothesize that adhesion and injury mediate signal transduction events causing the phosphorylation of tyrosine residues of specific adhesion proteins and that phosphorylation is required for cellular adhesion and migration. METHODS: For the adhesion experiments; primary rabbit stromal fibroblasts were seeded and phosphorylation of tyrosine residues was followed from 1 min to 24 h. For the injury experiments, confluent primary cultures were rendered quiescent, wounded, and tyrosine phosphorylation was followed from 30 s to 6 h. The antibody (py-20) was used to detect proteins phosphorylated on tyrosine residues. We examined changes in the phosphorylation of focal adhesion kinase (FAK), paxillin and cortactin, using immunoprecipitation and Western blot analysis. RESULTS: In the adhesion experiments, the phosphorylation of a 68-kDa protein was detected after 1 min, and the phosphorylation of a 125-kDa protein was not detected until 15 min. These proteins were identified in re-probed blots as paxillin and FAK. In the injury experiments, FAK phosphorylation was detected within 30 s and remained elevated for 6 h when cells were cultured on fibronectin. Both FAK and paxillin phosphorylation were prominent after injury, but unlike FAK phosphorylation, paxillin phosphorylation decreased over time. Phosphorylation was prominent at the wound margin. After wound closure, it returned to background levels. Tyrosine kinase inhibitors, genistein and herbimycin, decreased the number of adherent cells and altered the rate of cell migration after injury, compared to control (DMSO alone). CONCLUSION: The results indicate that injury and cell-matrix interaction mediate the phosphorylation of specific adhesion proteins and that phosphorylation is required for wound repair.     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

The immune-endocrine loop during aging: role of growth hormone and insulin-like growth factor-I

Why a primary lymphoid organ such as the thymus involutes during aging remains a fundamental question in immunology. Aging is associated with a decrease in plasma growth hormone (somatotropin) and IGF-I, and this somatopause of aging suggests a connection between the neuroendocrine and immune systems. Several investigators have demonstrated that treatment with either growth hormone or IGF-I restores architecture of the involuted thymus gland by reversing the loss of immature cortical thymocytes and preventing the decline in thymulin synthesis that occurs in old or GH-deficient animals and humans. The proliferation, differentiation and functions of other components of the immune system, including T and B cells, macrophages and neutrophils, also demonstrate age-associated decrements that can be restored by IGF-I. Knowledge of the mechanism by which cytokines and hormones influence hematopoietic cells is critical to improving the health of aged individuals. Our laboratory has recently demonstrated that IGF-I prevents apoptosis in promyeloid cells, which subsequently permits these cells to differentiate into neutrophils. We also demonstrated that IL-4 acts much like IGF-I to promote survival of promyeloid cells and to activate the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase). However, the receptors for IGF-I and IL-4 are completely different, with the intracellular beta chains of the IGF receptor possessing intrinsic tyrosine kinase activity and the alpha and gammac subunit of the heterodimeric IL-4 receptor utilizing the Janus kinase family of nonreceptor protein kinases to tyrosine phosphorylate downstream targets. Both receptors share many of the components of the PI 3-kinase signal transduction pathway, converging at the level of insulin receptor substrate-1 or insulin receptor subtrate-2 (formally known as 4PS, or IL-4 Phosphorylated Substrate). Our investigations with IGF-I and IL-4 suggest that PI 3-kinase inhibits apoptosis by maintaining high levels of the anti-apoptotic protein Bcl-2. The sharing of common activation molecules, despite vastly different protein structures of their receptors, forms a molecular explanation for the possibility of cross talk between IL-4 and IGF-I in regulating many of the events associated with hematopoietic differentiation, proliferation and survival.     

IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.     

mu-Opioids activate tyrosine kinase focal adhesion kinase and regulate cortical cytoskeleton proteins cortactin and vinculin in chick embryonic neurons

We have investigated the signal transduction pathway of the G-protein mu-opioid receptor upstream of phospholipase D (PLD) and protein kinase C-epsilon (PKC-epsilon) activation in postmitotic E6CH chick embryo cortical neurons. The mu-opioid receptor and PLD-PKC-epsilon functional coupling depends on upstream tyrosine kinase activation. We now report that the mu-opioid agonists specifically stimulated tyrosine phosphorylation and activation of the focal adhesion kinase (FAK) in a time-dependent manner. We also demonstrate that met-enkephalin, a mu-opioid agonist in E6CH cultures, significantly increases tyrosine phosphorylation of another Src kinase substrate, the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin led to drastic changes in subcellular localization, an estimated 2-fold enrichment in the cytosol. Similarly, opioids stimulated a sustained tyrosine phosphorylation of vinculin, a protein enriched in focal adhesion sites. These data provide novel evidence that opioid receptor intracellular signaling engages the specific activation of tyrosine kinase FAK and regulates the neuronal cytoskeleton during central nervous system morphogenesis.     

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.     

Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.     

Signaling through scaffold, anchoring, and adaptor proteins

The process by which extracellular signals are relayed from the plasma membrane to specific intracellular sites is an essential facet of cellular regulation. Many signaling pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine residues of target proteins. Recently, it has become apparent that regulatory mechanisms exist to influence where and when protein kinases and phosphatases are activated in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks or by placing enzymes close to their substrates is discussed.     

Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1

Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of beta2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the beta2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of beta2 integrins at least partially through cytohesin-1.