Platelet-derived Growth Factor Stimulates Src-dependent mRNA Stabilization of Specific Early Genes in Fibroblasts (original) (raw)
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Proceedings of the National Academy of Sciences, 1993
Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microiajected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGFstimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell
PubMed, 2000
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.
Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene
Proceedings of the National Academy of Sciences, 2000
Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF-and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF-and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.
Oncogene, 1999
Activation of the platelet-derived growth factor (PDGF) receptor tyrosine kinase induces tyrosine phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. Since the PDGF receptor also activates the Src tyrosine kinase, it is possible that Src mediates tyrosine phosphorylation of STATs in PDGFtreated cells. Consistent with a role for Src in STAT activation, we found that a PDGF receptor juxtamembrane tyrosine residue required for Src activation is necessary and sucient for activation of STATs 1 and 3. To test the Src requirement further, we made other mutations in the PDGF receptor juxtamembrane region that increased or decreased Src binding. In epithelial and ®broblast cells, PDGF activated STAT1, 3 and 6 in the absence of detectable binding and activation of Src. In addition, PDGF induced c-myc RNA expression and DNA synthesis even though Src was not detectably activated. The activation of MAP kinase and the induction of c-fos gene expression both correlated with STAT but not Src activation by the receptor. We conclude that juxtamembrane tyrosine phosphorylation is necessary for both Src tyrosine kinase and STAT activation by the bPDGF receptor, but that both processes are regulated independently by this region.
Requirement of Phosphatidylinositol-3 Kinase for Activation of JNK/SAPKs by PDGF
Biochemical and Biophysical Research Communications, 1997
pholipase A2, p90 Rsk and nuclear proteins such as the The molecular mechanism by which cell surface reternary complex factor p62 TCF and ElK-1 (2). In conceptors stimulate the serine/threonine kinase activity trast, JNKs phosphorylate the amino terminal transof c-Jun N-terminal kinases (JNKs) was investigated activating domain of c-jun and ATF2 (3), thereby inusing a transient cotransfection experiments in COScreasing their transcriptional activity. 7 cells. Our data demonstrate that JNK activity is po-Although, recent findings have helped to unveil the tently induced by platelet derived growth factor pathway linking cell surface receptors to ERKs, the (PDGF) upon expression of bPDGFR wild type mechanism of activation of JNKs by receptor tyrosine (bRWT). However, PDGF failed to mediate JNK activakinases (RTKs) is less well defined (1,4). Platelet-detion in cells expressing bPDGFR mutant lacking the rived growth factor (PDGF) is a potent mitogen for binding site for phosphatidylinositol-3 (PI-3) kinase connective tissue cells (5). PDGF mediates its diverse but not for phospholipase Cg (PLCg) or Syp. Consisbiological functions by binding to two related high aftent with this result, a PI-3 kinase inhibitor, wortmanfinity receptors designated as aPDGFR and bPDGFR nin inhibited activation of JNK by PDGF. Further-(6,7). Activation of the PDGFR tyrosine kinase domain more, overexpression of P110 the catalytic domain of leads to physical association and tyrosine phosphory-PI-3 kinase was sufficient for activation of JNKs which could be efficiently inhibited by dominant negative lation of many substrates, including Src family memforms of Ras, Rac but not of RhoA or Cdc42. Taken bers Src, yes and fyn, the 85 kDa subunit of PI-3 kitogether all of these findings suggest that activation nase (p85), Nck, RasGTPase-activating protein (Rasof JNK by PDGF involves receptor association with PI-GAP), phospholipase C-g (PLCg) and Syp (8-13). 3 kinase activity, which in turn acts on a ras-and rac-The specific tyrosine residues interacting with each dependent pathway.
2009
Src non-receptor tyrosine kinase is an important component of the platelet-derived growth factor (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Srcfamily kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates-Calpain 2, Eps15 and Trim28-in a c-Src-dependent fashion.
PI3K is involved in PDGF-β receptor upregulation post-PDGF-BB treatment in mouse HSC
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2006
Increased expression of PDGF-β receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-β receptor by cathepsin B, thus suggesting that the absence of PDGF-β receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-β receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibit...
Biochemical Journal, 2000
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 plateletderived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF β-receptor (PDGF β-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF β-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