Taxol mediates serine phosphorylation of the 66-kDa Shc isoform

In the human lung carcinoma cell line: A549, Taxol (20 nM) causes a decreas ed electrophoretic mobility of the 66-kDa Shc isoform (p66shc), beginning 4 h after drug exposure, and reaching a maximum at 9-18 h, No shift was obse rved for the 52- and 46-kDa isoforms of Shc. The electrophoretic mobility s hift of p66shc caused by Taxol is not the result of tyrosine phosphorylatio n, and there is no indication of a Shc/Grb2 complex in Taxol-treated A549 c ells. This modification is blocked by the serine/threonine protein phosphat ase 2A. In vivo P-32-labeling and subsequent phosphoamino acid analysis of p66shc indicated that both the original and the shifted p66shc were predomi nantly serine phosphorylated. Cyanogen bromide digestion of p66shc produced a phosphorylated fragment with an apparent molecular weight of similar to 7.9 kDa from the untreated cells and two phosphorylated fragments, of simil ar to 7.9 and similar to 9.6 kDa, from the Taxol-treated cells. The domain of Taxol-induced serine phosphorylation is thought to be in the cyanogen br omide fragment containing residues 2-65. The Taxol induced electrophoretic mobility shift of p66shc was inhibited by the protein synthesis inhibitor, cycloheximide, but not by the mitogen-activated and extracellular signal-re gulated protein kinase kinase (MEK) inhibitor, PD98059, This mobility shift did not occur in Taxol-resistant A549-T12 cells treated with 20 nM Taxol. In addition to Taxol, other microtubule-interacting drugs caused a decrease d electrophoretic mobility of p66shc, This Taxol-mediated serine phosphoryl ation seen in p66shc may result from a MEK-independent signaling pathway th at is activated in cells that have a prolonged or abnormal mitotic phase of the tell cycle and may play a role in signaling events that lead to cell d eath.