Graduate Thesis Or Dissertation
 

Involvement of myristoylated alanine-rich c kinase substrate (MARCKS) protein in prostaglandin F2α- induced secretion of oxytocin by the bovine corpus luteum

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  • Prostaglandin F₂α (PGF₂α), a stimulatory hormone of luteal oxytocin secretion, is known to activate protein kinase C (PKC); however, the intracellular signals that promote exocytosis of oxytocin remain to be elucidated. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by PKC, crosslinks actin filaments associated with the inner leaflet of the plasma membrane. Studies were conducted to determine the role of the MARCKS protein in exocytosis of PGF₂α-induced bovine luteal oxytocin. Experiment 1 was conducted to examine the regulatory aspects and localization of MARCKS in the bovine corpus luteum (CL) in response to PGF₂α stimulation. Luteal cells were incubated with [32PJ-orthophosphate and stimulated with ethanol, PGF₂α, TPA and A23187. Treatments with PGF₂α, TPA and A23187 resulted in increased phosphorylation of MARCKS. Subsequently, heifers were injected with either saline or PGF2a and CL was collected 5 mm after treatments. Western blotting of the luteal samples indicated that PGF2- induced translocation of MARCKS from the membrane to cytoplasm within 5 mm. The aim of experiment 2 was to identify the specific PKC isoform activated by PGF₂α that phosphorylates MARCKS protein. Using isoform specific inhibitory peptides and polyclonal antibodies, it was observed that PKCx was the mediator of PGF₂α-induced phosphorylation of MARCKS. Experiment 3 was conducted to determine if phosphorylation dependent movement of MARCKS is related to the disruption of the actin cortex and exocytosis of oxytocin. For this purpose, luteal cells were transfected with green fluorescent protein (GFP) conjugated MARCKS cDNA constructs. Expression of MARCKS-GFP was observed within 18 hr after transfection. Cells were then treated with vehicle or PGF₂α and fixed with 4% paraformaldehyde. Cells were subjected to oxytocin antibody+rhodamine labeled secondary antibody and phalloidin, a specific marker of the actin cortex. Upon PGF₂α treatment, the wt MARCKS-GFP translocated from membrane to cytoplasm and actin filaments shortened. Oxytocin granules were mobilized towards the membrane from a paranuclear location. Phosphorylation and myristoylation mutant MARCKS-GFPs were not affected by PG₂α stimulation of cells and no exocytosis of oxytocin occurred. In summary, the research suggests that PGF₂α-induced PKCα mediated phosphorylation of MARCKS is closely correlated with exocytosis of oxytocin by the bovine CL.
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