The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGFactivated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass ( z 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/ polyacrylamide gel electrophoresis revealed further that the NGF-or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of x 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2 -3 h.The PC12 pheochromocytoma cell line is a useful system for studying the mechanism of action of nerve growth factor (NGF). Exposure of PC12 cells to NGF results in differentiation of the cells, i.e. the conversion from a chromaffinlike phenotype into a sympathetic-neuron-like phenotype [I]. Although the detailed biochemical mechanisms of action of NGF are not known, it is well known that treatment with NGF induces the enhanced phosphorylation of a number of proteins in PC12 cells [2 -81.The mechanisms of mitogenic signal transduction evoked by mitogens such as epidermal growth factor (EGF) has been intensively investigated so far, and a phosphorylation cascade is thought to play an essential role in transduction ofmitogenic signals [9, 101. MAP kinase is one of the kinases involved in the phosphorylation cascade 111 -141. We have previously found that treatment with various mitogens including EGF, platelet-derived growth factor, fibroblast growth factor, insuCorrespondence to E. Nishida,