Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser(338), MEK1/2 at Ser(217) and Ser(221), and ERK1/2 at Thr(202) and Tyr(204). Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.     

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.     

Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.     

Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.     

Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.

The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

CSF-1 stimulated multiubiquitination of the CSF-1 receptor and of Cbl follows their tyrosine phosphorylation and association with other signaling proteins

Addition of colony stimulating factor-1 (CSF-1) to macrophages stimulates the rapid, transient tyrosine phosphorylation, membrane association and multiubiquitination of Cbl (Wang et al. [1996] J. Biol. Chem. 271:17-20). Kinetic analysis reveals that the tyrosine phosphorylation of Cbl is coincident with its plasma membrane translocation and association with the activated tyrosine phosphorylated CSF-1 R, p85, Grb2, and tyrosine phosphorylated p58Shc and that these events precede the simultaneous multiubiquitination of Cbl and the CSF-1 R. Tyrosine phosphorylation and multiubiquitination of the cell surface CSF-1 R are stoichiometric and the multiubiquitinated CSF-1 R is degraded. Similarly, the membrane associated Cbl is almost stoichiometrically ubiquitinated, but the ubiquitinated Cbl is not degraded, being recovered, deubiquitinated, in the cytosol 3-10 min after stimulation at 37 degrees C. In the membrane fraction of cells stimulated at 4 degrees C, the association of p58Shc and Grb2 with Cbl is stable, whereas its association with Sos and p85 is transient and their dissociation occurs at the time CSF-1 R and Cbl multiubiquitination commence. The membrane translocation and the pattern of association of Sos with the CSF-1R, p85, Grb2, and p58Shc resemble those of Cbl but Sos is not tyrosine phosphorylated, nor multiubiquitinated and the coprecipitation of these proteins, other than Grb2, with Sos is much less. Complexes formed by Sos and Cbl are largely independent and membrane complexes of Cbl with other tyrosine phosphorylated proteins, p85 and Grb2 also contain CSF-1 R. These data raise the possibility that the predicted negative regulatory role of Cbl in macrophages is its enhancement of ligand-induced CSF-1 R internalization/degradation.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Regulation of tyrosine phosphorylation in macrophage phagocytosis and chemotaxis

Macrophages display a large variety of surface receptors that are critical for their normal cellular functions in host defense, including finding sites of infection (chemotaxis) and removing foreign particles (phagocytosis). However, inappropriate regulation of these processes can lead to human diseases. Many of these receptors utilize tyrosine phosphorylation cascades to initiate and terminate signals leading to cell migration and clearance of infection. Actin remodeling dominates these processes and many regulators have been identified. This review focuses on how tyrosine kinases and phosphatases regulate actin dynamics leading to macrophage chemotaxis and phagocytosis.     

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.     

Ubiquitylation of MEKK1 inhibits its phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways

MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways.     

Endothelin-1 inhibits adipogenesis: role of phosphorylation of Akt and ERK1/2

In adipogenesis, growth factors play a crucial role. Using serum depleted condition, we studied the causal role of endothelin-1 (ET-1) and epidermal growth factor (EGF), separately or together, in adipocyte differentiation of 3T3-L1 cells. ET-1 stimulation caused an anti-adipogenic response and this effect was potentiated upon treatment with EGF. Co-treatment with EGF and ET-1 blocked the expression of C/EBPalpha and PPARgamma, the adipogenic markers. The inhibition of adipogenesis was preceded by a biphasic (early and late) attenuation of Akt phosphorylation. We suggest that treatment with ET-1 and EGF together induce a more potent anti-adipogenic response, involving increased Erk1/2 phosphorylation and biphasic attenuation of Akt phosphorylation.     

Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-factor-induced Rac activation

The non-receptor tyrosine kinase Abl participates in receptor tyrosine kinase (RTK)-induced actin cytoskeleton remodelling, a signalling pathway in which the function of Rac is pivotal. More importantly, the activity of Rac is indispensable for the leukaemogenic ability of the BCR-Abl oncoprotein. Thus, Rac might function downstream of Abl and be activated by it. Here, we elucidate the molecular mechanisms through which Abl signals to Rac in RTK-activated pathways. We show that Sos-1, a dual guanine nucleotide-exchange factor (GEF), is phosphorylated on tyrosine, after activation of RTKs, in an Abl-dependent manner. Sos-1 and Abl interact in vivo, and Abl-induced tyrosine phosphorylation of Sos-1 is sufficient to elicit its Rac-GEF activity in vitro. Genetic or pharmacological interference with Abl (and the related kinase Arg) resulted in a marked decrease in Rac activation induced by physiological doses of growth factors. Thus, our data identify the molecular connections of a pathway RTKs-Abl-Sos-1-Rac that is involved in signal transduction and actin remodelling.     

Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity

Depending on the cellular context, Ras can activate characteristic effectors by mechanisms still poorly understood. Promotion by galectin-1 of Ras activation of Raf-1 but not of phosphoinositide 3-kinase (PI3-K) is one such mechanism. In this report, we describe a mechanism controlling selectivity of K-Ras4B (K-Ras), the most important Ras oncoprotein. We show that galectin-3 acts as a selective binding partner of activated K-Ras. Galectin-3 co-immunoprecipitated significantly better with K-Ras-GTP than with K-Ras-GDP, H-Ras, or N-Ras and colocalized with green fluorescent protein-K-Ras(G12V), not with green fluorescent protein-H-Ras(G12V), in the cell membrane. Co-transfectants of K-Ras/galectin-3, but not of H-Ras/galectin-3, exhibited enhanced and prolonged epidermal growth factor-stimulated increases in Ras-GTP, Raf-1 activity, and PI3-K activity. Extracellular signal-regulated kinase (ERK) activity, however, was attenuated in K-Ras/galectin-3 and in K-Ras(G12V)/galectin-3 co-transfectants. Galectin-3 antisense RNA inhibited the epidermal growth factor-stimulated increase in K-Ras-GTP but enhanced ERK activation and augmented K-Ras(G12V) transformation activity. Thus, unlike galectin-1, which prolongs Ras activation of ERK and inhibits PI3-K, K-Ras-GTP/galectin-3 interactions promote, in addition to PI3-K and Raf-1 activation, a third inhibitory signal that attenuates active ERK. These experiments established a novel and specific mechanism controlling the duration and selectivity of signals of active K-Ras, which is extremely important in many human tumors.     

Contortrostatin activates ERK2 and tyrosine phosphorylation events via distinct pathways

We report that cells adhering to contortrostatin show transient increases in activation of Extracellular signal Regulated Kinase 2 (ERK2). The kinetics and degree of activation are similar to cells adhering to fibronectin or vitronectin. We have recently shown that contortrostatin induces tyrosine phosphorylation in tumor cells. Contortrostatin is shown here to stimulate activation of ERK2 in suspended cells, but this activation follows a different dose-response pattern than contortrostatin-induced tyrosine phosphorylation. Since contortrostatin induces tyrosine phosphorylation via alphavbeta3, we explored the effects of an alphavbeta3-blocking antibody, 7E3, on contortrostatin-stimulated ERK2 activation. While 7E3 completely blocks the effect of contortrostatin on tyrosine phosphorylation, this antibody had no effect on activation of ERK2. In cells lacking expression of alphavbeta3, tyrosine phosphorylation was unaffected by contortrostatin treatment, but ERK2 was activated. This is strong evidence that contortrostatin is regulating tyrosine phosphorylation events and ERK2 activation via separate pathways and through different integrin receptors.