Minako Hoshi

Protein kinase C phosphorylates tau and induces its functional alterations.

We found that tau, one of the major microtubule‐associated proteins, is a good substrate for protein kinase C. The phosphorylation occurred mainly on serine residues and the sites phosphorylated by protein kinase C were largely different from those phosphorylated by cAMP‐dependent protein kinase as analyzed by phosphopeptide mapping. The protein kinase C‐mediated phosphorylation of tau reduced its abilities to promote tubulin polymerization and to cross‐link actin filaments. The reduction in its abilities was in proportion to the number of phosphates incorporated into tau.

ACTIVATION OF MICROTUBULE-ASSOCIATED PROTEIN KINASE BY MICROTUBULE DISRUPTION IN QUIESCENT RAT 3Y1 CELLS

Treatment of quiescent rat fibroblastic cells (3Y1) with colchicine, a microtubule-disrupting agent, which could induce the initiation of DNA synthesis [Y. Shinohara, E. Nishida, and H. Sakai (1989) Eur. J. Biochem. 183, 275-280], activated a serine/threonine-specific protein kinase activity in cell extracts that preferentially phosphorylated exogenous microtubule-associated protein 2 (MAP2). Vinblastine treatment also activated the kinase activity, and taxol pretreatment inhibited the colchicine-induced activation of this kinase activity. The detailed biochemical characterization indicated that this microtubule disruption-activated MAP2 kinase was very similar or identical to the mitogen-activated MAP kinase in the substrate specificity and chromatographic behaviors on phosphocellulose, DEAE-cellulose, gel filtration, and phenyl-Sepharose. Pretreatment of the cells with protein synthesis inhibitors did not prevent the MAP2 kinase activation by colchicine. Moreover, phosphatase treatment inactivated the colchicine-activated MAP2 kinase activity. These data suggest that microtubule disruption activates MAP kinase through phosphorylation.

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.

Activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein by mitogenic stimuli in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate (TPA) activated MAP kinase, whereas progression factors such as insulin did not. Insulin was, however, capable of activating MAP kinase in cells pretreated with TPA. Moreover, TPA plus insulin activated MAP kinase more strongly and for a longer time period than did TPA alone. Treatment of Balb/c 3T3 cells with competence factors stimulated phosphorylation of the 350-kDa protein which was immunoprecipitated with antibodies against brain high-molecular-weight microtubule-associated protein MAP1, whereas insulin treatment did not stimulate the phosphorylation. Insulin could induce, however, further increase in the phosphorylation of the 350-kDa protein, when added simultaneously with TPA or added to the TPA-treated cells. The enhanced phosphorylation of the 350-kDa protein thus correlated with the MAP kinase activation. As insulin acts synergistically with TPA to induce initiation of DNA synthesis in the quiescent Balb/c 3T3 cells, it seems that activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein are accompanied by the initiation of DNA synthesis.

Glucose and nitrogen rapidly activate a Ca2+‐inhibitable, serine/threonine kinase activity toward microtubule‐associated protein 2 in Saccharomyces cerevisiae

Treatment of the glucose‐starved yeast cells (Saccharomyces cerevisiae) with 1% glucose or 2‐deoxyglucose induced a rapid increase in a protein kinase activity in cell extracts that phosphorylated microtubule‐associated protein 2 (MAP2) in vitro. Addition of 0.5% ammonium sulfate to the nitrogen‐starved yeast cells also stimulated the kinase activity toward MAP2. The stimulated MAP2 kinase activities had the following common properties: (i) Activation was rapid and transient in response to stimuli; (ii) The kinase activity was serine/threonine‐specific; and (iii) The kinase activity was inhibited by micromolar concentrations of free Ca2+. These properties are very similar to those of the mitogen‐activated, Ca2+‐sensitive MAP2 kinase we have recently found in mammalian fibroblastic cells. The MAP2 kinase activation may be involved in initiation of proliferation of yeast cells.