Correlation between protein phosphorylation and progesterone synthesis in bovine luteal cells stimulated by lutropin.

Abstract

The implication of cytosolic protein phosphorylation in the regulation by lutropin of steroidogenesis in luteal tissue was investigated, in vitro by incubating selected small bovien luteal cells in the presence of 32P‐labelled phosphate. The incorporation of 32P into cytosolic proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and autoradiography of the gel. Progeserone synthesis, 32P incorporation into cytosolic proteins, cyclic AMP content and protein kinase activity were measured simultaneously aafter incubation for various times and with increasing doses of lutropin.As previously reporte, the gonadotropin as well as dibutyryladenosine 3′, 5′ monophosphate enchanced by 60–150% the degree of phosphorylation of six cytosolic proteins of molecular weight ranging from 50000–115000.Time‐course studies in the presence of a saturating dose of lutropin demonstrated a close correlation between progesterone synthesis, protein phosphorylation and cyclic AMP production. The three parameters were increased within 5 min, the level of cylci AMP and the extent of protein phosphorylation reaching a maximum value after 10‐20 min.Dose‐response curves for progesterone synthesis and protein phosphorylation were nearly identical with a half‐maximal stimulation occurring for both parameters with a physiological concentration of lutropin (3ng/ml). Conversely dose‐response curves for cyclic AMP and protein kinase activity were clearly shifted, as expected, toward the higher concentration of lutropin (half‐maximal stimulation for 90 ng/ml of lutropin).Further characterization of the phosphorylated proteins using a highly resolutive two‐dimensional eletrophoretic technique demonstrated that the number of proteins phosphrylated under lutropin and the degree of stimulation was distinctly higher than suspeted from monodimensional fractionation technique. The 32P incorporation fro two of the nine proteins phosphorylated by lutropin appeared at least ten‐times higher than in the control.The present result are compatible with the implication of at least some of the observed unduced protein phosphorylations in the steroidogenic effect of lutropin in luteal tissue. They sustain the hypothesis of a cyclic‐AMP‐dependent protein kinase activation as a part of the mechanism of lutropin action.