Tyrosine Phosphorylation of Gsα and Inhibition of Bradykinin-induced Activation of the Cyclic AMP Pathway in A431 Cells by Epidermal Growth Factor Receptor

Abstract

An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (G s␣ There is mounting evidence indicative of complex, probably cell-specific interactions between signaling pathways involving heterotrimeric G proteins and tyrosine kinases. For example, the stimulation of G protein-coupled receptors modulates key proteins of the mitogen-activated protein kinase pathway via protein kinase C-or protein kinase A-mediated phosphorylation on serine or threonine residues (1-3). Furthermore, several isoforms of ␣ subunits of G proteins were shown to be phosphorylated in vitro on tyrosine residues by tyrosine kinase receptors such as the epidermal growth factor (EGF) 1 receptor and the insulin receptor or by non-receptor tyrosine kinases of the Src kinase family. EGF was shown to activate cardiac adenylate cyclase via a mechanism requiring both G s␣ and the EGF receptor tyrosine kinase (4, 5). EGF was also found to stimulate phospholipase C in rat hepatocytes (6) or phospholipase A 2 in rat kidney (7) in a pertussis toxin-sensitive manner, suggesting an involvement of G i proteins. The molecular mechanism of these receptor tyrosine kinase-mediated effects remained unclear. In reconstituted phospholipid vesicles, the insulin receptor was found to catalyze tyrosine phosphorylation of G i␣ and G o␣ , suggesting the possibility of a direct interaction of receptor tyrosine kinases and G proteins (8). Further progress in this field came from in vitro studies of Hausdorff et al. (9) on direct interactions between the non-receptor tyrosine kinase pp60 c-src and purified G protein ␣ subunits. pp60 c-src was shown to phosphorylate recombinant G s␣ as well as other G ␣ isoforms on tyrosine residues almost stoichiometrically (9). In 1995, the in vitro sites of phosphorylation of G s␣ by pp60 c-src were identified (10). The phosphorylated tyrosine residues are located at N-terminal position 37 and at C-terminal position 377 of G s␣ and thus in regions known to participate in nucleotide exchange and receptor interaction (10, 11). Very recently, using purified EGF receptor and recombinant G s␣ , Poppleton et al. (12) demonstrated an activation of G s␣ via phosphorylation on tyrosine residues by EGF receptor kinase. However, little is known about tyrosine phosphorylation of G s␣ in intact cells and how such phosphorylation might affect the function of the G protein. We have recently shown that bradykinin (BK) activates dual pathways in A431 human epidermoid carcinoma cells, i.e. the phospholipase C-␤/protein kinase C pathway and, independently via G s␣ , the cyclic AMP/protein kinase A pathway, which represents a negative feedback loop to the BKinduced protein kinase C activation (13). At the same time, as observed earlier by...

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.…”

Epidermal growth factor receptor tyrosine kinase inhibition is not protective in PCK rats

“…EGFR tyrosine kinase activation triggers numerous downstream signaling pathways, including phosphorylation of G proteins, activation of phospholipase C, and downstream calcium and protein kinase C (PKC)-mediated cascades, and stimulation of the ERK pathway [52]. Purified EGFR can phosphorylate G sa in vitro [53] and in vivo [54] and directly stimulate of adenylyl cyclase; at the same time, it prevents G sa activation by G protein-coupled receptors, such as the bradykinin receptor, and reduces cAMP accumulation [54]. An increase in intracellular Ca 2+ , such as occurs following EGFR activation, can stimulate cAMP phosphodiesterases, inhibit adenylyl cyclases, and inhibit vasopressin-induced renal accumulation of cAMP [55].…”

A Novel Mitogenic Signaling Pathway of Bradykinin in the Human Colon Carcinoma Cell Line SW-480 Involves Sequential Activation of a Gq/11 Protein, Phosphatidylinositol 3-Kinase β, and Protein Kinase Cε

“…Among the PTX-insensitive G proteins expressed in SW-480 cells, G 12/13 do not stimulate phosphatidylinositol hydrolysis (36) and may be excluded from linking the bradykinin receptor to phospholipase C␤. The bradykinin receptor appears to be capable of interacting with multiple G proteins, including also G s (23,37). If the effect of bradykinin on MAPK is triggered by ␤␥-complexes released from a G s protein as demonstrated for the ␤-adrenergic receptor (9), it might be expected that permanent activation of G s in the presence of CTX simulates or potentiates the BK action on MAPK.…”