Abstract: 218

PHOSPHORYLATION OF MARCKS PROTEIN IN MEMBRANE AND CYTOPLASM OF BOVINE LUTEAL CELLS.

U Salli¹ , SA Supancic¹ , F Stormshak^{1 *}
Departments of Biochemistry and Animal Sciences, Oregon State University, Corvallis, OR ¹

Effect of known activators of PKC on intracellular distribution of phosphorylated myristoylated alanine rich C kinase substrate (MARCKS) protein in bovine corpora lutea (CL) on Day 8 of the estrous cycle was examined. CL from heifers (n=4) were subjected to collagenase to obtain a heterogenous population of luteal cells. Cells (10 × 10⁶) were placed into each of 4 flasks containing 2 ml of phosphate deficient medium and incubated at 37°C for 1.5 h. After this period, [³²P]-orthophosphate (100 Ci/ml) was added to each flask and the incubation continued for 1.5 h. Cells were then exposed to the following agents for 30 min: 1) ethanol (control), 2) PGF₂ (56 nM), 3) phorbol ester (TPA, 1M) and 4) Ca²⁺ ionophore A23187 (5 M). Cells were homogenized, centrifuged and membrane and cytosolic fractions heated to permit recovery of heat stable MARCKS. Cell fractions were subjected to 7.5% SDS-PAGE and the proteins were transferred for Western Blot analysis. Blots were autoradiographed and protein bands exposed by use of monoclonal MARCKS antibody. MARCKS on film and blots were quantified by densitometry (D). Exposure of cells to PGF₂, TPA and A23187 stimulated (p<0.01) phosphorylation of membrane-bound MARCKS (control, 0.42±0.17; PGF₂, 2.86±1.91; TPA, 3.89±1.99 and A23187, 2.66±1.11 ³²P-D units/D unit of blot protein) and phosphorylation of cytosolic MARCKS (control, 1.44±0.47; PGF₂, 4.95±1.55; TPA, 5.95±1.45 and A23187, 4.41±1.58 ³²P-D units/D unit of blot protein. These data suggest that activation of PKC results in phosphorylation of MARCKS protein in luteal cells without affecting a change in membrane to cytosolic ratio of the protein. Supported by USDA NRI Grant 97-35203-4681.

    This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.