PHOSPHORYLATION OF A 27-KDA PROTEIN CORRELATES WITH SURVIVAL OF PROTEIN-SYNTHESIS-INHIBITED MCF-7 CELLS

Previously, we have shown that IGF-1, the phorbol ester 12-0-tetradeca noylphorhol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protect ed MCF-7 cells against death induced by the protein synthesis inhibito r cycloheximide (CHX). We proposed that phosphorylation of a putative cellular protein(s) map be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to t heir survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-depe ndent manner in CHX-treated MCF-7 cells. The ED(50) values observed we re 25 ng/ml, 40 mu g/ml and 15 ng/ml for TPA, ATA, and IGF-1, respecti vely. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuou sly to control levels. The 27-kDa protein was found in the cytosolic f raction as a phosphorylated serine residue. Further characterization w ith two-dimensional electrophoresis indicated that the 27-kDa phosphop rotein was resolved into two isoforms with pi 5.7 and 5.9. Such charac teristics were observed for the small molecular weight heat shock prot ein HSP27. Indeed, a single band of 27 kDa was detected immunologicall y with rabbit polyclonal anti-human HSP27. The inactive phorbol ester alpha TPA, epidermal growth factor (EGF), and 8-bromoadenosine 3'5'-cy clic monophosphate (Br-cAMP) did not increase phosphorylation of the 2 7-kDa protein. Cell survival was measured by exposure of the CHX-pretr eated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas alpha TPA, EGF, and Br-cAMP did not. Our results indicate a correlati on between phosphorylation of a 27-kDa protein, probably HSP27, and en hanced cell survival, suggesting a role fdr this phosphoprotein in the survival mechanism.