Abstract. We previously demonstrated (Ookata et al., 1992(Ookata et al., , 1993 that the p34~¢2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34 ~dc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anticyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34~d~Vcyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34~cVcyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34 ~c2, cyclin B, and the COOHterminal domain of MAP4, Phu, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34 ~d~2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.p ROGaESSION through M-phase of the cell cycle is controlled by M-phase promoting factor (MPF) i, which consists of a complex of p34 cd~2 and cyclin B (for

Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1 . Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26-and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5 þ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22 þ (a Swi2/Snf2-ADCR homologue) deletion and snf22 þ gcn5 þ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

A novel method to observe the autolytic activation of a mammalian cytoplasmic calcium protease, mu-calpain, was developed using a set of antipeptidic antibodies capable of distinguishing between the pre- and post-autolysis forms of the enzyme. Antibodies raised against synthetic peptides designed to match the N-terminal sequences of the pre- and post-autolysis forms of the mu-calpain large subunit reacted specifically with the corresponding form of calpain and not with the other. The antibodies were specific and sensitive enough to detect the antigens in crude cell lysates. The relevance of the immunochemical detection of calpain activation was confirmed by the observation that proteolysis of a substrate protein by purified mu-calpain paralleled autolysis at various pCa as probed by these antibodies and that autolysis preceded substrate proteolysis. We also observed calcium-dependent autolysis of calpain accompanying subsequent proteolysis of substrate in intact cells using the antibodies. The method will provide a novel approach to assess the physiological targets of the enzyme by determining the local intracellular sites of calpain activation.