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 different signaling systems in a clonal strain of murine Leydig tumor cells (designated MA-10), both compounds ultimately elicit several common effects, such as increased steroidogenesis, decreased transcription of the LH/CG receptor gene, and attenuation of adenylyl cyclase. Based on this information, it was hypothesized that hCG and mEGF may induce phosphorylation of a common protein or set of proteins. Studies were performed to identify such a protein if it exists. MA-10 cells were metabolically labeled with [32P]orthophosphate, and phosphorylation patterns were examined and quantitated using two-dimensional gel electrophoresis. A 21-kilodalton protein whose phosphorylation state increased after hCG and mEGF stimulation was identified. Three isoforms of this protein were visible after hCG stimulation, with approximate pI values of 5.9, 6.0, and 6.1, while only two isoforms were visible after mEGF stimulation, with approximate pI values of 6.0 and 6.1. This protein is phosphorylated on serine residues in response to either hCG or mEGF. Since this protein may play a role in eliciting the common actions of hCG and mEGF, its time course of phosphorylation and concentration responsiveness to hCG and mEGF were also characterized.