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]P(i). TPA induced a characteristic pattern of labelled proteins, four in 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 α and β subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca²⁺ of 0.1 μM and 2 μM. For both forms of PKC, in particular the β 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 β 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.