Protein kinase A and C activities and endogenous substrates in ovine small and large luteal cells

Abstract

Protein kinase A (cAMP-dependent) and C (calcium, phospholipid-dependent) activities were measured and in vitro phosphorylation of endogenous proteins by these kinases were observed by SDS-PAGE in 100000 × g supernatant (soluble) fractions of ovine small (12–22 μm) and large (> 22 μm) luteal cells. No differences in stimulation ( P < 0.05) of A kinase activity between small and large cells were detected. Protein kinase C activity was stimulated ( P < 0.05) 2.9-fold in small cells but not significantly enhanced above basal ( P > 0.05) in large cells. By direct comparison, greater stimulation ( P < 0.05) over basal of A versus C kinase (6.1- versus 2.9-fold) was measured in small cells. These stimulations were greater than those observed in large cells (A kinase, 4.8-fold; C kinase, 1.8-fold). Maximal specific activities of both kinases (per mg protein) were greater ( P < 0.05) in small than in large cells. Endogenous proteins that could serve as substrates for phosphorylation by A and C kinases differed between small and large cells. Phosphorylation of six proteins by A kinase was consistently greater in small than in large cells. One endogenous protein (37 kDa) appeared to serve as a preferred substrate for phosphorylation by A kinase in small cells and C kinase in large cells. One protein (81 kDa) was predominantly phosphorylated in large rather than small cells by a calcium-dependent, C kinase-indepen- dent mechanism. These results support the accepted role of cAMP via A kinase and a possible role for C kinase in regulating Steroidogenesis in ovine small luteal cells. The inability of large cells to respond to cAMP with enhanced secretion of progesterone may be due to an unavailability of phosphoprotein substrates for A kinase. Furthermore, protein kinase C activity and available protein substrates display quantitative and qualitative differences between small and large cells. Differences in regulation of Steroidogenesis between the cell types may be due to these observed differences.