Mitogen-induced phosphorylation of human B-lymphocyte proteins. Relationship to protein kinase C activation.
We have investigated the rapid phosphorylation of proteins in B-lymphocytes incubated with the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), anti-Ig and combinations of TPA and the Ca2+ ionophore ionomycin. Two-dimensional electrophoretic analysis was used to identify the proteins phosphorylated in cells preincubated with [32P]Pi. TPA induced a characteristic pattern of labelled proteins, four of which (pp85, pp76, pp66 and pp63) showed a dose-dependent incorporation of 32P on serine residues. The phosphorylation of pp63 and pp66, in particular, correlated with the mitogenic dose-response curve. Addition of the Ca2+ ionophore ionomycin to B-cells also stimulated a characteristic incorporation of 32P into proteins, which included pp63 and pp66. With combined doses of TPA and ionomycin, these two proteins show an enhanced phosphorylation, which correlated well with the synergistic enhancement of proliferation shown by this combination of agents. Protein kinase C (PKC) was partially purified from B-cells and separated into alpha and beta subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca2+ of 0.1 microM and 2 microM. For both forms of PKC, in particular the beta form, higher concentrations of Ca2+ shifted the dose-response curve for TPA to the left and increased the maximum activation. Anti-Ig, which stimulated B-cells by cross-linking surface immunoglobulin and causing hydrolysis of PtdIns(4,5)P2, also caused increased phosphorylation of several proteins, which again included pp63 and pp66. These data suggest that PKC, particularly the beta form, is involved in the early part of the proliferation cascade for human B-lymphocytes. It is most probably activated in a synergistic manner by the increased Ca2+ and diacylglycerol levels which result from the earlier hydrolysis of PtdIns(4,5)P2.
Guy
G R
GR
Department of Biochemistry, University of Birmingham, U.K.
Finney
M
M
Michell
R H
RH
Gordon
J
J
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis; D004357:Drug Synergism; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects; D008934:Mitogens / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003432:Cross-Linking Reagents; D008934:Mitogens; D011506:Proteins; D015759:Ionomycin; D004247:DNA; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate