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.

Effects of phorbol esters on an interleukin-3-dependent cell line.

FDC-P1 is an interleukin-3 (IL-3)-dependent cell line that ceases to proliferate in the absence of IL-3. We have isolated variant cell lines from FDC-P1 that grow in response to phorbol myristate acetate (PMA). These variant cell lines (FD/PMA) have maintained their PMA-dependency for over 1 year. Lymphokine gene expression, which would support growth, was not detected in FD/PMA lines. FD/PMA lines had a different cell surface phenotype than the parental line. Mac-1, Mac-2, and Mac-3 were readily detected on the cell surface of FD/PMA lines; however, these antigens were not detected on FDC-P1. Because protein kinase C (PKC) activation may mediate PMA effects, we examined this kinase. PKC activity quantitated by 32P-incorporation into histone was increased in FDC-P1 as compared with FD/PMA cultured in IL-3. Moreover, PKC activity was undetectable in FD/PMA lines cultured in PMA. Using Western blotting, immunoreactive PKC was readily detected in cytosolic and solubilized particulate fractions of FDC-P1 cells, not but in FD/PMA cell extracts. Comparisons between the parental and FD/PMA lines should provide insight into IL-3- and PMA-mediated signal transduction.