FORMATION OF A DISTINCT CONNEXIN43 PHOSPHOISOFORM IN MITOTIC CELLS ISDEPENDENT UPON P34(CDC2) KINASE

The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacr ylamide gel electrophoresis. The electrophoretic mobility of connexin4 3 from mitotic cells was distinctly reduced to a form (P3) that migrat ed slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-t reated and untreated cultures. Mitotic FT210 cells, which contain a te mperature-sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 maintained at the connexin43 when permissive temperat ure where p34(cdc2) is active. In contrast, nocodozole-treated FT210 c ells grown at the nonpermissive temperature did not contain P3 connexi n43. These results indicated that generation of the P3 connexin43 was dependent upon active p34(cdc2)/cyclin B kinase. Although the p34(cdc2 ) kinase phosphorylated connexin43 in vitro on peptides containing ser ine 255, the major phosphotryptic peptides in P3 connexin43 from mitot ic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase. The P3 connex in43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures, The se events may be part of the dramatic structural changes observed in m itotic cells undergoing cell rounding and cytokinesis. Results of init ial studies using inhibitors of protein degradative and synthetic path ways suggested the likelihood that protein degradation and synthesis p articipate in the disappearance of the P3 connexin43 and restoration o f the pattern of connexin43 isoforms observed in nonmitotic cells.