PROTEIN-KINASE-C PHOSPHORYLATES P50 LSP1 AND INDUCES TRANSLOCATION OFP50 LSP1 IN T-LYMPHOCYTES

A lymphocyte-specific protein, p50, is phosphorylated on Ser and Thr r esidues in mitogen-activated T cells, suggesting that this molecule pl ays some role in the T cell activation cascade, p50 was identified as lymphocyte specific protein 1 (LSP1), which is a putative calcium-bind ing protein. In the present study, to clarify the role of p50 protein in the cascade, in vivo and in vitro phosphorylation of this molecule, and the effect of the phosphorylation on its distribution in activate d T cells were examined. First, to obtain a sufficient amount of p50 a s a phosphorylation substrate, p50 cDNA, which encodes a protein of 33 0 amino acid residues with a molecular mass of 36,728 Da, was cloned f rom an ICR mouse thymocyte cDNA library and expressed in Escherichia c oil. When the putative coding region of p50 cDNA was expressed in E. c oli, the product showed an apparent molecular mass of 50 kDa on SDS-PA GE. The recombinant p50 was phosphorylated in vitro by rabbit protein kinase C (PKC) and by murine cytosolic protein kinase, that was activa ted by a combination of phosphatidylserine and diacylglycerol. Further more, p50 was shown to be phosphorylated on the same sites in T cells upon stimulation with Con A as when phosphorylated in vitro by rabbit PHC, indicating that p50 is phosphorylated by PKC in Con A-stimulated T cells. On subcellular fractionation followed by immunoblotting analy sis, membrane-bound p50 was shown to be released from the membrane fol lowing activation of PKC in T cells. These results and the recent find ing that p50 binds to actin fibers raise the possibility that p50 cont rols the binding of actin fibers to the plasma membrane under regulati on by PKC in T cells.