Phorbol ester- and luteinizing hormone-induced phosphorylation of membrane proteins in bovine luteal cells.
In this study we have investigated the protein phosphorylation pattern in the membrane fraction prepared from bovine luteal cells. The phosphorylation reaction was carried out in vitro, under defined conditions, using either [gamma-32P]ATP or [gamma-35S]ATP as the phosphate donor. The results obtained show that [gamma-35S]ATP was a suitable phosphate donor for performing in vitro phosphorylation studies, and that thiophosphorylation of at least eight protein bands (120 kDa to 18 kDa) was observed. The extent of phosphorylation was dependent upon the duration of incubation and the amount of membrane protein used. The presence of Ca2+ was obligatory for phosphorylation and an enhanced phosphorylation was observed in the presence of Ca2+, phosphatidyl serine and phorbol 12-myristate 13-acetate (PMA), agents known to activate protein kinase C. Interestingly, when phosphorylation was carried out in the presence of luteinizing hormone (LH), a phosphorylation pattern was obtained which was similar to that obtained in the presence of calcium and phospholipid. Furthermore, in the case of two protein bands corresponding to 80-82 and 44-46 kDa, an additive phosphorylation was observed when the phosphorylation reaction was carried out for 5 min in the presence of both LH and Ca2+, phosphatidyl serine and PMA. To conclude, we have demonstrated a calcium- and phospholipid-dependent endogenous protein phosphorylation in the membrane fraction prepared from bovine luteal cells and the data obtained suggest that LH is able to stimulate this endogenous protein phosphorylation via a protein kinase C-mediated mechanism.
Budnik
L T
LT
Institute for Hormone and Fertility Research, Hamburg, F.R.G.
Mukhopadhyay
A K
AK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003338:Corpus Luteum / Q000378:metabolism*; D005260:Female; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D008565:Membrane Proteins; D010718:Phosphatidylserines; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D007986:Luteinizing Hormone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium