Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes.
Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.
Unit of Applied Cell and Molecular Biology, University of Umeå, Sweden.
M
M
Gullberg
K
K
Noreus
G
G
Brattsand
B
B
Friedrich
V
V
Shingler
D000595:Amino Acid Sequence; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology*
D010446:Peptide Fragments; D010710:Phosphates; D010750:Phosphoproteins; D011506:Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C