Protein kinase C phosphorylates p50 LSP1 and induces translocation of p50 LSP1 in T lymphocytes.
A lymphocyte-specific protein, p50, is phosphorylated on Ser and Thr residues in mitogen-activated T cells, suggesting that this molecule plays some role in the T cell activation cascade. p50 was identified as lymphocyte specific protein 1 (LSP1), which is a putative calcium-binding 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 activated T cells were examined. First, to obtain a sufficient amount of p50 as a phosphorylation substrate, p50 cDNA, which encodes a protein of 330 amino acid residues with a molecular mass of 36,728 Da, was cloned from an ICR mouse thymocyte cDNA library and expressed in Escherichia coli. When the putative coding region of p50 cDNA was expressed in E. coli, the product showed an apparent molecular mass of 50 kDa on SDS-PAGE. The recombinant p50 was phosphorylated in vitro by rabbit protein kinase C (PKC) and by murine cytosolic protein kinase, that was activated by a combination of phosphatidylserine and diacylglycerol. Furthermore, 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 PKC, indicating that p50 is phosphorylated by PKC in Con A-stimulated T cells. On subcellular fractionation followed by immunoblotting analysis, membrane-bound p50 was shown to be released from the membrane following activation of PKC in T cells. These results and the recent finding that p50 binds to actin fibers raise the possibility that p50 controls the binding of actin fibers to the plasma membrane under regulation by PKC in T cells.
Matsumoto
N
N
Department of Parasitology, Institute of Medical Science, University of Tokyo.
Kojima
S
S
Osawa
T
T
Toyoshima
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D005815:Genetic Code; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000097:blood; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000378:metabolism*
D018076:DNA, Complementary; D010750:Phosphoproteins; D011994:Recombinant Proteins; C093915:lymphocyte-specific protein p50; D011493:Protein Kinase C