Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654.

Abstract

Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of '25I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol.diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with (32liphosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P~phosphoserine and [32P~phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654, previously shown to be phosphorylated in response to phorbol diesters in intact cells or by purified protein kinase C in vitro. Further, it was observed that PDGF mimicked the action of phorbol diesters to inhibit the EGF-dependent tyrosine phosphorylation of the EGF receptor in [32Pjphosphate-labeled fibroblasts.These results are consistent with the hypothesis that increases in diacylglycerol and Ca2' levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.Platelet-derived growth factor (PDGF) is a potent polypeptide mitogen that is released into the blood during platelet lysis after tissue injury. The full mitogenic effect of PDGF is only expressed in the presence of other growth factors such as epidermal growth factor (EGF) and insulin (1). Interestingly, this effect is also characteristic of tumor-promoting phorbol diesters such as 4p-phorbol 12/3-myristate 13a-acetate (PMA), which stimulate the growth of quiescent fibroblasts synergistically with EGF and insulin (2, 3). An-other striking similarity between the actions of PDGF and phorbol diesters is a rapid inhibition of 125I-labeled EGF (125I-EGF) binding to the receptors ofcultured cells (4-12). In view of the similarity of these actions of PDGF and PMA on the EGF receptor, we considered the possibility that these two agents may have a similar mechanism of action.It has been reported that PMA causes the phosphorylation of the EGF receptor on senine and threonine residues, and it has been suggested that this phosphorylation is causally related to the effect of PMA on the EGF receptor (13)(14)(15). Phosphopeptide mapping of the EGF receptor has revealed that the phosphorylation state of several sites is increased after treatment of cells with phorbol diester (13,15,16). One of these sites was observed to be uniquely phosphorylated on the EGF receptor isolated from PMA-trea...

Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction

“…2B). This is an expected result, given that FGF causes transmodulation of EGF receptors, a phenomenon whereby activation of heterologous receptors, such as platelet-derived growth factor and bFGF receptors, results in phosphorylation and desensitization of the EGF receptor (3,17,21,27). In bFGF-desensitized cells, we observed little, if any, effect on anisomycin-stimulated induction of fosB, c-jun, junB, and junD ( Fig.…”

“…Heterologous desensitization can also occur at the level of receptors. For example, platelet-derived growth factor and bFGF activate serine/threonine kinases (ceramideactivated protein kinase [36] and protein kinase C [reference 27 and references therein]) that phosphorylate the EGF receptor, resulting in their conversion from high to low affinity (transmodulation [3,17,21]). This has been characterized in C3H 10T 1 ⁄2 cells (27) and may contribute to partial desensitization of EGF-inducible IE genes in bFGF-pretreated cells.…”

The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase.

“…Finally, it seems more likely that removal of phosphate from the serine and threonines in the constitutively phosphorylated EGF receptor would stimulate kinase activity. Most of the same sites that are constitutively phosphorylated on the EGF receptor are enhanced in their phosphorylation after treatment of cells with tumor promoters, which results in inhibition of kinase activity (11,16,31).…”

Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo.

“…This suggested a second difference in regulation between normal and transforming p185, since normal p185 expressed at low levels in Rat-1 cells does not contain phosphotyrosine. Although the increased tyrosine phosphorylation of pl85* expressed at low levels might simply mirror a greater activity of the pl85* kinase, it may be relevant to the mechanism of transformation by p185 since other tyrosine kinases are regulated by tyrosine phosphorylation (5,6,8,10,25,(29)(30)(31) (16,22; but see references 7 and 44), but the EGF receptor is not phosphorylated on tyrosine when expressed at low levels (12,13). (In these experiments there are no direct comparisons of high-and low-level EGF receptor expression, so it is difficult to be certain that tyrosine phosphorylation of the EGF receptor expressed at low levels would be detected.)…”