EGF receptor‐mediated, c‐Src‐dependent, activation of Stat5b is downregulated in mitogenically responsive hepatocytes

Abstract

Signal transducer and activator of transcription (STAT) proteins may be activated by epidermal growth factor (EGF), but their role in EGF receptor-mediated mitogenic signaling is not clear. We previously showed that Stat5b was activated by EGF in rat hepatocytes in primary monolayer culture. In the present study, we found that EGF induced tyrosine phosphorylation of Stat5b both on Tyr-699, which correlated with specific DNA binding activity, and also on other tyrosine residues. The Src tyrosine kinase inhibitor CGP77675 blocked the EGF-induced activation of Stat5b, but did not affect the Stat5b activation by growth hormone (GH) or prolactin (PRL). The Stat5b response to EGF was most pronounced soon (3 h) after plating (early G 1 ) and at high cell density (50,000 hepatocytes per cm 2 ). However, at this cell density EGF did not stimulate DNA synthesis. In hepatocytes at 24 h of culturing (mid/late G 1 ) with 20,000 cells per cm 2 , EGF induced strong phosphorylation of the EGF receptor, as well as Shc and ERK, and stimulated DNA synthesis, but did not activate Stat5b, although the Stat5b response to GH or PRL was retained. A strong GHinduced Stat5b activation neither influenced the DNA synthesis alone nor enhanced the mitogenic effect of EGF. The results show that EGF induces tyrosine phosphorylation and DNA-binding activity of Stat5b in a manner different from GH and PRL, apparently by a Src-dependent mechanism. The data also provide further evidence that Stat5b is not required for mitogenic signaling from the EGF receptor.

SOCS2 Induces Neurite Outgrowth by Regulation of Epidermal Growth Factor Receptor Activation

“…4). As reported previously (28,29), this effect of EGF on STAT5 activation was at least partly due to Src signaling. Addition of PP2 inhibited STAT5 phosphorylation in control cells, although it did not further decrease STAT5 phosphorylation in SOCS2-PC12 cells (Fig.…”

“…In hepatocytes from SOCS2Ϫ/Ϫ mice, STAT5b phosphorylation in response to GH was moderately prolonged, and the overgrowth phenotype of the SOCS2Ϫ/Ϫ mice was abrogated in the absence of STAT5b (27). STAT5b has also recently been shown to be activated downstream of EGFR signaling in a c-Src-dependent manner in fibroblasts and breast tumor cell lines (28) and in hepatocytes (29). However, STAT5 phosphorylation and activation by EGF are quantitatively and qualitatively different from that induced by GH, which phosphorylates STAT5b at Tyr 699 .…”

“…However, STAT5 phosphorylation and activation by EGF are quantitatively and qualitatively different from that induced by GH, which phosphorylates STAT5b at Tyr 699 . Analysis of STAT5 using anti-phosphotyrosine antibodies has shown that EGF induces higher maximal levels of total STAT5 phosphorylation than GH, but that if an antibody to STAT5b phosphorylated on Tyr 699 was used, then the EGFinduced phosphorylation was lower than GH-induced phosphorylation in hepatocytes (29), indicating that sites other than…”

Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis

“…Furthermore, STAT5b activated by c-Src displays a unique nuclear localization pattern as seen by immunofluorescence, when compared to the pattern of STAT5b activated by prolactin, again suggesting a novel function (Kabotyanski and Rosen, 2003). Additional evidence of a unique model of EGFR/c-Src-induced activation was recently described in a rat hepatocyte model system (Guren et al, 2003). Here, EGF-induced activation of STAT5b not only results in a unique tyrosine phosphorylation pattern of STAT5b, but its activation by EGF is dependent on c-Src kinase activity.…”

The interplay between Src family kinases and receptor tyrosine kinases

“…Stats are activated by a variety of peptide mitogens (Bromberg and Darnell, 2000). Several different studies have used both pharmacological inhibitors and dominant-negative forms of SFKs, and concluded that SFKs are required for both PDGF (Blake et al, 2000;Wang et al, 2000;Bowman et al, 2001;Simon et al, 2002) and EGF (Olayioye et al, 1999;Kazansky and Rosen, 2001;Guren et al, 2003;Kloth et al, 2003) stimulation of Stat tyrosine phosphorylation and activation. The nonreceptor tyrosine kinase Pyk2 is also involved in the SFKmediated activation of Stats in response to growth factors (Shi and Kehrl, 2004), although the mechanism by which this occurs is not clear.…”