MPM-2 ANTIBODY-REACTIVE PHOSPHORYLATIONS CAN BE CREATED IN DETERGENT-EXTRACTED CELLS BY KINETOCHORE-BOUND AND SOLUBLE KINASES

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated ph osphoproteins, The major phosphoproteins of mitotic chromosomes recogn ized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared b y detergent lysis in the presence of potent phosphatase inhibitors, th e MPM-2 antibody labels phosphoproteins found at kinetochores, chromos ome arms, midbody and spindle poles of mitotic cells, In cells extract ed without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished, However, in cytoskeletons this ep itope can be regenerated through the action of kinases stably bound at the kinetochore, Various kinase inhibitors mere tested in order to ch aracterize the endogenous kinase responsible for these phosphorylation s, We found that the MPM-2 epitope will not rephosphorylate in the pre sence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the reph osphorylation indicating that the endogenous MPM-2 kinase at kinetocho res is not p34(cdc2), casein kinase II, MAP kinase, protein kinase A o r protein kinase C. The addition of N-ethylmaleimide inactivated the e ndogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34(cdc2)- cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosophoepitope, However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores, Furthermor e, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are dist inct, Our results suggest that multiple kinases (either soluble or kin etochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope, The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in k ey events such as mitotic kinetochore assembly and sister chromatid se paration at anaphase.