Activation of Gsα by the Epidermal Growth Factor Receptor Involves Phosphorylation

Abstract

Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates G s␣ and activates this protein. Employing purified EGFR and G s␣ , we have demonstrated that the EGFR kinase phosphorylates G s␣ in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of G s␣ . As determined by phosphoamino acid analysis, the phosphorylation of G s␣ by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5-3-O-(thio)triphosphate (GTP␥S) inhibited the phosphorylation of G s␣ without altering EGFR autophosphorylation. However, G protein ␤␥ subunits protected against GDP-and GTP␥S-mediated inhibition of phosphorylation of G s␣ . In functional studies, phospho-G s␣ demonstrated a greater GTPase activity and also a greater capacity to bind GTP␥S as compared to the nonphosphorylated G s␣ . Moreover, the phospho-G s␣ augmented adenylyl cyclase activity in S49 cyc ؊ cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of G s␣ on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase. Epidermal growth factor (EGF)1 exerts a variety of biological actions ranging from increased DNA synthesis, hyperplasia, and increased glucose and fatty acid metabolism, to alterations in muscular function (see Ref. 1 for review). These pleiotropic actions of EGF are mediated via the activation of several second messenger systems. For instance, following binding of EGF to its receptors, the intrinsic protein tyrosine kinase activity of the EGF receptor is increased, resulting in autophosphorylation of the EGF receptor as well as of other cellular proteins (reviewed in Refs. 1 and 2). The autophosphorylation of the EGF receptor serves to recruit proteins containing the Src homology 2 (SH2) domains such as phospholipase C␥ (3), the subsequent phosphorylation of which increases phosphatidylinositol metabolism, and the generation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (3). Likewise, recruitment of the SH2-containing proteins such as Grb2 and other adaptor proteins to the phosphotyrosine-containing domains on the EGF receptor also leads to the activation of serine/threonine phosphorylation cascades such as the mitogen-activated protein kinase cascade (4). In addition, EGF has also been documented to modulate the cAMP second messenger system. Studies from our laboratory have shown that EGF increases contractility, beating rate, and cAMP accumulation in the heart (5) by stimulating adenylyl cyclase via a process involving G s␣ (6, 7). Moreover, the protein tyrosine kinase activity of the EGF receptor is important for EGF-mediated stimulation of cardiac adenylyl cyclase (8). One implication of this latter finding is that one, or more, of the signaling elements involved in stimulation of adenylyl cyclase by the activated EGF receptor is(are) phosphorylated. Therefore, we have proposed the hyp...

Divergent requirement for Gαs and cAMP in the differentiation and inflammatory profile of distinct mouse Th subsets

“…Moreover, the cAMP-induced Ca 2+ influx and subsequent Th17 differentiation were PKA dependent ( Figure 7). Previous data have suggested that Gαs can directly activate Ca 2+ channels (28,29) and non-receptor and receptor tyrosine kinases (30)(31)(32); however, our results indicating that addition of a cAMP analog could reverse the phenotype of the Gnas ΔCD4 CD4 + T cells imply that the effects of Gαs on Th subset differentiation is likely to be mediated, at least for the most part, by decreased cAMP accumulation in these cells.…”

Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2

“…#P Ͻ 0.001 compared with asbestosexposed siControl. the tyrosine kinase activity of EGFR is required for growth factor stimulation of adenylyl cyclase activity, which leads to the activation of PKA (17,21). In our study, we found that CREB phosphorylation following crocidolite asbestos exposure was reduced by inhibiting PKA and EGFR activity.…”

Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric Gαs Protein

“…Alternatively, activation of the EGF receptor by EGF could trigger the translocation of G␣s. It has been shown that G␣s is tyrosine phosphorylated by the EGF receptor in vitro and in response to EGF stimulation in vivo (Liebmann et al, 1996;Poppleton et al, 1996). Conceivably, this phosphorylation event might be related to the change in the subcellular localization of G␣s.…”