MITOGENIC RESPONSE OF MURINE B-LYMPHOCYTES TO SALMONELLA-TYPHIMURIUM LIPOPOLYSACCHARIDE REQUIRES PROTEIN-KINASE C-DEPENDENT LATE TYROSINE PHOSPHORYLATIONS

Unlike the cross-linking of membrane immunoglobulins, the activation o f B cells by lipopolysaccharide (LPS) does not involve the phosphoinos itol turnover and the initial activation of tyrosine kinases, However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture. Tyrosyl-phosphorylated proteins were detecte d by Western blotting after 24 h of culture with LPS, reaching a maxim um concentration after 72 h, Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M anti body and were abrogated by the protein synthesis inhibitor cycloheximi de, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine. The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+ mobilizati on and was confirmed by detection of a comparable but restricted patte rn of tyrosine-phosphorylated substrates in B cells treated with phorb ol myristate acetate alone or in association with ionomycin, Tyrosine kinase activation was dependent on de novo protein synthesis. However, culture supernatants of LPS-activated B cells were devoid of mitogeni c activity and induced a phosphorylation pattern more restricted than that achieved by LPS, Altogether these data indicate that proliferatio n signals induced by LPS or by the cross-linking of membrane immunoglo bulins are controlled by late tyrosine phosphorylations occurring thro ughout the first 3 days of culture, controlled in part by protein kina se C activation, and dependent on the synthesis of an intermediate pro tein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.