Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells

Abstract

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.

Prolactin modulates phosphorylation, signaling and trafficking of epidermal growth factor receptor in human T47D breast cancer cells

“…In these cells, both molecules were basally tyrosine phosphorylated, as evidenced by their detection with anti-pTyr antibody (middle panels of Figure 2a and b, lane 1), suggesting a level of activation of each independent of added EGF. Notably, PRL treatment did not increase basal tyrosine phosphorylation of EGFR in T47D cells (Figure 2a, middle panel, lane 2 vs 1), unlike previous findings for GH in other cell systems (Yamauchi et al, 1997;Huang et al, 2003). ErbB-2 tyrosine phosphorylation was decreased by PRL treatment (Figure 2b, middle panel, lane 2 vs 1), consistent with our previous findings for GH's effects on ErbB-2 in preadipocytes (Kim et al, 1999).…”

“…Eluates were resolved by SDS-PAGE and immunoblotted sequentially with PTP101 (upper panels), anti-pTyr (middle panels) and anti-EGFR or anti-ErbB-2 (lower panels) ( Figure 2a and b, respectively). As we previously detected for GH stimulation in 3T3-F442A cells (Kim et al, 1999;Huang et al, 2003), PRL acutely promoted PTP101-reactive phosphorylation of both EGFR and ErbB-2 (upper panels of Figure 2a and b, respectively, lane 2 vs 1) in T47D cells. In these cells, both molecules were basally tyrosine phosphorylated, as evidenced by their detection with anti-pTyr antibody (middle panels of Figure 2a and b, lane 1), suggesting a level of activation of each independent of added EGF.…”

“…We previously reported that in mouse preadipocytes GH acutely induced threonine phosphorylation of both EGFR and ErbB-2, as detected by a monoclonal antibody (PTP101) that recognizes phosphorylation of the consensus sites in the substrates for proline-directed kinases such as ERKs (Huang et al, 2003). We similarly examined the effects of PRL on the phosphorylation status of EGFR and ErbB-2 in T47D cells (Figure 2).…”

“…Recent studies suggest that PRL, growth hormone (GH) and other cytokines may use multiple mechanisms to crosstalk with EGFR and/or ErbB-2 (Johnson et al, 1996;Fenton and Sheffield, 1997;Wiepz et al, 1997;Yamauchi et al, 1997Yamauchi et al, , 1998Yamauchi et al, , 2000Qiu et al, 1998;Quijano and Sheffield, 1998;Kim et al, 1999;Maus et al, 1999;Badache and Hynes, 2001;Huang et al, 2003). For example, GH causes EGFR tyrosine phosphorylation without the activation of EGFR kinase activity (Yamauchi et al, 1997;Kim et al, 1999).…”

“…In that case, GH-induced tyrosinephosphorylated EGFR binds Grb2 and enhances GH-induced ERK activation. GH also causes ERKmediated threonine phosphorylation of EGFR and ErbB-2 (Kim et al, 1999;Huang et al, 2003). The effect of GH-induced EGFR threonine phosphorylation in murine preadipocytes is to dampen subsequent EGFinduced EGFR downregulation, augmenting signaling of the activated receptor (Huang et al, 2003).…”