IDENTIFICATION OF A LEYDIG TUMOR-CELL PROTEIN THAT IS PHOSPHORYLATED IN RESPONSE TO STIMULATION WITH CHORIOGONADOTROPIN OR EPIDERMAL GROWTH-FACTOR

Although hCG and mouse epidermal growth factor (mEGF) activate differe nt signaling systems in a clonal strain of murine Leydig tumor cells ( designated MA-10), both compounds ultimately elicit several common eff ects, such as increased steroidogenesis, decreased transcription of th e LH/CG receptor gene, and attenuation of adenylyl cyclase. Based on t his information, it was hypothesized that hCG and mEGF may induce phos phorylation of a common protein or set of proteins. Studies were perfo rmed to identify such a protein if it exists. MA-10 cells were metabol ically labeled with [P-32]orthophosphate, and phosphorylation patterns were examined and quantitated using two-dimensional gel electrophores is. A 21-kilodalton protein whose phosphorylation state increased afte r hCG and mEGF stimulation was identified. Three isoforms of this prot ein were visible after hCG stimulation, with approximate pl values of 5.9, 6.0, and 6.1, while only two isoforms were visible after mEGF sti mulation, with approximate pI values of 6.0 and 6.1. This protein is p hosphorylated on serine residues in response to either hCG or mEGF. Si nce this protein may play a role in eliciting the common actions of hC G and mEGF, its time course of phosphorylation and concentration respo nsiveness to hCG and mEGF were also characterized.