Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.
Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.
Tombes
R M
RM
Laboratory of Molecular Biology, University of Wisconsin, Madison 53706.
Peloquin
J G
JG
Borisy
G G
GG
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D016213:Cyclins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D006656:Protamine Kinase / Q000378:metabolism*; D008941:Spindle Apparatus / Q000201:enzymology*; D013379:Substrate Specificity
D016213:Cyclins; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; C085284:microtubule-associated protein 1B; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase