Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line.
FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O-tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine-independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.
Ways
D K
DK
Department of Medicine, East Carolina University School of Medicine, Greenville, NC 27858-4354.
Qin
W
W
Riddle
R S
RS
Garris
T D
TD
Bennett
T E
TE
Steelman
L S
LS
McCubrey
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D015153:Blotting, Western; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D002999:Clone Cells; D007377:Interleukin-3 / Q000494:pharmacology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000276:immunology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000906:Antibodies; D007377:Interleukin-3; D007527:Isoenzymes; D010455:Peptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate