RESUMO
The bovine corpus luteum contains a myristoylated alanine-rich C kinase substrate (MARCKS) protein known to crosslink actin filaments in the cytoskeletal cortex associated with the plasma membrane. We conducted experiments to determine whether concentrations of MARCKS mRNA and protein in the bovine corpus luteum varied during the estrous cycle. Using Northern blots probed with a MARCKS cDNA, we found that luteal concentrations of MARCKS mRNA were greatest on d 4, 8, and 12 and markedly reduced on d 16 of the cycle (p < 0.08). Similarly, Western blot analysis of luteal proteins revealed that concentrations of MARCKS protein were greatest on d 8 and least on d 16 of the cycle (p < 0.01). Exposure of slices from a d 8 corpus luteum to prostaglandin F2alpha (PGF2alpha) during a 10-min incubation in the presence of [32P]-ortho-phosphate resulted in enhanced phosphorylation of MARCKS in membrane and cytosolic fractions compared to that of controls. We therefore concluded that expression of the luteal MARCKS protein gene may be regulated and that PGF2alpha-induced phosphorylation of this protein is attributable to activation of protein kinase C.
Assuntos
Corpo Lúteo/química , Estro , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas/análise , Proteínas/genética , RNA Mensageiro/análise , Animais , Northern Blotting , Western Blotting , Bovinos , Membrana Celular/metabolismo , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Citosol/metabolismo , Dinoprosta/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Substrato Quinase C Rico em Alanina Miristoilada , Fosfatos/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Fatores de TempoRESUMO
The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.
