PHOSPHORYLATION OF ANNEXIN-XI (CAP-50) IN SR-3Y1 CELLS

Annexin XI (CAP-50) is a probable target protein of calcyclin. Being d ifferent from other annexins, annexin XI localizes mainly in nuclei of cultured fibroblasts. In rat embryonic fibroblasts transformed by Rou s sarcoma virus oncogene, SR-3Y1 cells, phosphorylation of annexin XI was increased on both serine and threonine residues (Ser < Thr), compa red with findings in control 3Y1 cells. The amount of phosphorylated a nnexin XI was approximately 8.5% of the total cellular annexin XI and the phosphorylated annexin XI migrated slightly slower on SDS-polyacry lamide gel electrophoresis than did the non-phosphorylated form of ann exin XI. Phosphorylated annexin XI was recovered in the cytoplasmic fr action and did not bind to phosphatidylserine vesicle in the presence of high Ca2+ (over 1 mM). Annexin XI was phosphorylated by mitogen-act ivated protein (MAP) kinase, which was reported to be activated in v-s rc-transformed fibroblast (Gupta, S. K., Gallego, C. Johnson, G. L. an d Heasley, L. E. (1992) J. Biol. Chem. 267, 7987-7990), on both serine and threonine residues (Ser >> Thr) in vitro. Comparative phosphopept ide mappings analyzed by reverse-phase high performance liquid chromat ography suggested that the sites phosphorylated in situ in SR-3Y1 cell s are distinct from the sites by MAP kinase. Annexin XI phosphorylated by MAP kinase still possessed the ability to bind to phosphatidylseri ne vesicle. These results suggest that annexin XI is a substrate for s ome Ser/Thr kinase(s) which is activated in v-src-transformed cells an d that the phosphorylation may regulate the function of annexin XI in living cells.