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Identification of a Leydig tumor cell protein that is phosphorylated in response to stimulation with
1Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242.
Abstract:
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.
Insights
Human chorionic gonadotropin (hCG) and mouse epidermal growth factor (mEGF) stimulate a common 21-kDa protein in MA-10 Leydig cells. This protein
Area of Science:
- Endocrinology
- Molecular Cell Biology
Background:
- Human chorionic gonadotropin (hCG) and mouse epidermal growth factor (mEGF) trigger distinct signaling pathways in MA-10 Leydig cells.
- Despite different pathways, both hCG and mEGF induce common cellular effects, including increased steroidogenesis and decreased luteinizing hormone/chorionic gonadotropin (LH/CG) receptor gene transcription.
Purpose of the Study:
- To investigate the hypothesis that hCG and mEGF converge on a common protein phosphorylation event.
- To identify and characterize a potential common protein substrate for hCG and mEGF signaling.
Main Methods:
- MA-10 Leydig cells were metabolically labeled with [32P]orthophosphate.
- Two-dimensional gel electrophoresis was employed to analyze and quantify protein phosphorylation patterns.
- Phosphorylation of specific proteins in response to hCG and mEGF stimulation was examined.
Main Results:
- A 21-kilodalton (kDa) protein was identified, exhibiting increased phosphorylation upon stimulation by both hCG and mEGF.
- hCG stimulation resulted in three isoforms of the 21-kDa protein (pI ~5.9, 6.0, 6.1), while mEGF stimulation showed two isoforms (pI ~6.0, 6.1).
- This 21-kDa protein is phosphorylated on serine residues in response to both hCG and mEGF, with characterized time course and concentration responsiveness.
Conclusions:
- A common 21-kDa protein is phosphorylated by both hCG and mEGF in MA-10 Leydig cells.
- This protein's phosphorylation may mediate the shared cellular responses elicited by hCG and mEGF.
- Further characterization of this protein is warranted to elucidate its precise role in Leydig cell function.