THE CONSERVED MITOTIC KINASE POLO IS REGULATED BY PHOSPHORYLATION ANDHAS PREFERRED MICROTUBULE-ASSOCIATED SUBSTRATES IN DROSOPHILA EMBRYO EXTRACTS

The Drosophila gene polo encodes a protein kinase required for progres sion through mitosis. Wild-type polo protein migrates as a tight doubl et of 67 kDa which is converted to a single band by phosphatase treatm ent, which also inactivates the kinase. We have determined putative po lo substrates in a cell-free system derived from mutant embryos, Exoge nous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo(1)- derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endo genous kinases, Proteins of the same size are predominantly phosphoryl ated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo(1)-derived embryos, We show that a specific monoclonal antibody t o beta-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase, Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations, We discuss the extent to which these in viv o phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle.