Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor.
ABSTRACT: The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure-function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.
Project description:Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.
Project description:Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.
Project description:Lung cancer is a major disease carrying heterogeneous molecular lesions and many of them remain to be analyzed functionally in vivo. Gain-of-function (GOF) SHP2 (PTPN11) mutations have been found in various types of human cancer, including lung cancer. However, the role of activating SHP2 mutants in lung cancer has not been established. We generated transgenic mice containing a doxycycline (Dox)-inducible activating SHP2 mutant (tetO-SHP2(E76K)) and analyzed the role of SHP2(E76K) in lung tumorigenesis in the Clara cell secretory protein (CCSP)-reverse tetracycline transactivator (rtTA)/tetO-SHP2(E76K) bitransgenic mice. SHP2(E76K) activated Erk1/Erk2 (Erk1/2) and Src, and upregulated c-Myc and Mdm2 in the lungs of bitransgenic mice. Atypical adenomatous hyperplasia and small adenomas were observed in CCSP-rtTA/tetO-SHP2(E76K) bitransgenic mice induced with Dox for 2-6 months and progressed to larger adenoma and adenocarcinoma by 9 months. Dox withdrawal from bitransgenic mice bearing magnetic resonance imaging-detectable lung tumors resulted in tumor regression. These results show that the activating SHP2 mutant promotes lung tumorigenesis and that the SHP2 mutant is required for tumor maintenance in this mouse model of non-small cell lung cancer. SHP2(E76K) was associated with Gab1 in the lung of transgenic mice. Elevated pGab1 was observed in the lung of Dox-induced CCSP-rtTA/tetO-SHP2(E76K) mice and in cell lines expressing SHP2(E76K), indicating that the activating SHP2 mutant autoregulates tyrosine phosphorylation of its own docking protein. Gab1 tyrosine phosphorylation is sensitive to inhibition by the Src inhibitor dasatinib in GOF SHP2-mutant-expressing cells, suggesting that Src family kinases are involved in SHP2 mutant-induced Gab1 tyrosine phosphorylation.
Project description:We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr(139). Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr(139), and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2), is not phosphorylated at Tyr(139) following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr(139) of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr(139) in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P(3), and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr(139). These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr(139) by a Src-family tyrosine kinase.
Project description:Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress.
Project description:Activation of the mitogen-activated protein kinase pathway represented by extracellular signal-regulated kinases (ERK1/2) and activation of the upstream kinase (MEK1) are critical events for growth factor signal transduction. c-Src has been proposed as a common mediator for these signals in response to both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein 1 (GIT1) is a substrate for c-Src that associates with MEK1 in vascular smooth-muscle cells and human embryonic kidney 293 cells. GIT1 binding via coiled-coil domains and a Spa2 homology domain is required for sustained activation of MEK1-ERK1/2 after stimulation with angiotensin II and epidermal growth factor. We propose that GIT1 serves as a scaffold protein to facilitate c-Src-dependent activation of MEK1-ERK1/2 in response to both GPCRs and TKRs.
Project description:Objectives:Grb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, migration, and function from multiple tyrosine kinase receptors. This study was designed to investigate the influence of upregulation of Gab1 in endothelial progenitor cells (EPCs) stimulated with hepatocyte growth factor (HGF), and the underlying molecular mechanisms. Materials and Methods:Endothelial progenitor cells isolated from human umbilical cord blood were identified and divided into four groups. EPCs in the Control group were cultured normally; those in the Control+HGF group were treated with HGF stimulation; those in the AD-Gab1 group were transfected with adenovirus containing the Gab1 gene but not treated with HGF stimulation; and, those in the AD-Gab1+HGF group were treated with both HGF stimulation and transfection with adenovirus containing the Gab1 gene. Subsequently, Gab1 expression and proliferation and migration ability were compared for EPCs grown under different conditions. Furthermore, we measured phosphorylation levels of three key proteins Gab1, SHP2, and ERK1/2. Results:The AD-Gab1+HGF group had the highest expression of Gab1 and higher proliferation and migration than the other three groups. Conclusions:Upregulation of Gab1 promoted HGF-induced EPC proliferation and migration. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in EPCs, thus leading to activation of extracellular regulated MAP kinase 1/2, which is involved in proliferation and migration signaling.
Project description:Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF beta-receptor (PDGF beta-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF beta-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-beta-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr(579) and Tyr(581) in PDGF beta-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src(-/-) and Fyn(-/-) mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF beta-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.
Project description:The macrophage migration inhibitory factor (MIF) receptor (CD74) was cloned recently, but the signaling mechanism is not evident. We hypothesized that signaling requires an additional molecule such as CD44, which activates nonreceptor tyrosine kinases. We utilized the CD74- and CD44-deficient COS-7/M6 cell to create stable transfectants expressing CD74, CD44, and a truncated CD44 lacking its intracytoplasmic signaling domain. CD74 alone mediated MIF binding; however, MIF-induced ERK1 and ERK2 kinase phosphorylation required the coexpression of full-length CD44. MIF binding was associated with the serine phosphorylation of CD74 and CD44. Investigations that used siRNA or kinase inhibitors indicate that MIF-induced ERK1 and ERK2 activation through CD44 required the Src tyrosine kinase. Studies of CD74, CD44, and CD74-CD44 transformants and corresponding mutant cells showed that CD74 and CD44 were necessary for MIF protection from apoptosis. These data establish CD44 as an integral member of the CD74 receptor complex leading to MIF signal transduction.
Project description:Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.