Complex formation of Plk1 and INCENP required for metaphase-anaphase transition

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.     

Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.     

Inhibition of ethylene production by osmotic shock. Further evidence for membrane control of ethylene production

Effects of osmotic shock applied to mung bean hypocotyl segmentswere examined to see if auxin-induced ethylene production isregulated by membrane activity. Uptake of -aminoisobutyric acidwas significantly inhibited by osmotic shock. While cold shockalone also inhibited the uptake, cold osmotic shock was mosteffective. Auxin-induced ethylene production by the osmoticallyshocked tissue was similarly inhibited. Shock treatment of auxin-pretreatedtissue also suppressed the subsequent ethylene production. Oxygenuptake was not affected by the shock treatment. Sorbitol andsucrose used as osmotic substance were as effective as mannitol.The results suggest that ethylene production is controlled bymembrane activity. (Received July 18, 1977; )     

RNA synthesis in the early stage of aerobic incubation of potato tuber discs

RNA synthesis of potato tuber discs during the early periodof their aerobic incubation was investigated by feeding thediscs with ³H-uridine. The rate of total RNA synthesis increasedin two steps during the incubation. The increase during thefirst 2 to 3 hr was small, but that after 3 hr was large. Thelabeled RNAs were separated into poly(A) containing RNA [poly(A)(+) RNA] and poly(A) lacking RNA [poly(A) (–) RNA] bythe use of a poly(U)-Sepharose column. Poly(A) (+) RNA was synthesizedeven in the freshly prepared discs which incorporated little¹⁴C-leucine into a protein fraction, and the synthetic rateof poly(A) (+) RNA increased by about 50% during the first 3hr incubation period, then gradually decreased thereafter. Synthesisof poly(A) (–) RNA continued to increase up to 7 hr afterslicing. When the discs were pulse labeled, the proportion ofradioactivity in poly(A) (+) RNA to that in the total RNA wasmaintained at about 50% until about 3 hr after slicing, butit abruptly decreased between 3 and 5 hr to about 35% whichwas maintained up to 9 hr after slicing. (Received October 12, 1977; )     

Role of oxygen in auxin-induced ethylene production

Ethylene production by IAA-treated mung bean hypocotyl segmentsunder various oxygen levels in the ambient atmosphere was examined.Rate of ethylene production was dependent upon oxygen levels,and gave a sigmoidal curve against oxygen levels. Tissue segmentspreincubated with IAA in low oxygen levels (1–10% O₂ inN₂) produced ethylene without a lag period at a rate higherthan that by control tissue segments preincubated in air, whenthey were exposed to a high oxygen level (air, 21% O₂). Theeffect of cycloheximide on tissue segments transferred froma low oxygen level to air was not much different from that onethylene production by control tissue segments previously incubatedin air. Incorporation of U-¹⁴C-leucine into the protein fractionby tissue segments placed in nitrogen was negligible, but thatin 2% oxygen was 10 to 14% of that in air. It was concluded that oxygen was an essential factor for boththe induction process of the ethylene producing system and thesynthesis of ethylene, and that although synthesis of ethyleneis dependent upon oxygen levels, formation of the ethylene producingsystem proceeded even under low oxygen levels. (Received January 13, 1977; )     

Molecular Size of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from Cucurbita maxima Duch. and Change of Translatable mRNA of the Enzyme after Wounding

Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthasewas purified by an immunoaffinity column from wounded mesocarpof winter squash (Cucurbita maxima Duch. cv. Ebisu) fruit, anda specific antibody was raised in rabbit. Translatable mRNAcoding for ACC synthase was barely detectable in fresh tissuebut clearly increased after wounding. The apparent molecularsize of the purified enzyme as estimated by SDS-polyacrylamidegel electrophoresis (PAGE) was about 50 kDa. However, SDS-PAGEfluorograms of in vitro translation product of ACC synthasemRNA and the in vivo labeled enzyme as well as Western blotanalysis showed that the subunit size of the enzyme was 58 kDa.The enzyme was partially degraded or processed to a 50 kDa peptideboth in vivo and in vitro. (Received December 19, 1987; Accepted June 13, 1988)