Kazushi Tanabe

Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK)

Background: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments.

EVIDENCE THAT SER-82 IS A UNIQUE PHOSPHORYLATION SITE ON VIMENTIN FOR CA2+-CALMODULIN-DEPENDENT PROTEIN KINASE II

We identified the sites on vimentin that are phosphorylated by Ca2(+)-calmodulin-dependent protein kinase II (CaM-kinase II). Sequential analysis of the purified phosphopeptides demonstrated that the sites are -Thr-Arg-Thr-Tyr-Ser(PO4)38-Leu-Gly-Ser-Ala- and -Val-Arg-Leu-Leu-Gln-Asp-Ser(PO4)82-Val-Asp-, which are located within the amino-terminal head domain of vimentin. For Ser-82 but not Ser-38, the proposed CaM-kinase II recognition amino acid sequence (Arg-X-X-Ser/Thr) was not found. Studies with a series of synthetic peptide analogs corresponding to Ser-82 and its surrounding amino acid sequence indicate that Asp-84 acts as an essential substrate specificity determinant for the Ser-82 phosphorylation by CaM-kinase II. The CaM-kinase II recognition site may be more extensive than heretofore determined.

Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomycespombe

Summary The gene for the large subunit of glutathione synthetase (EC 6.3.2.3) of Schizosaccharomyces pombe was cloned from a S . pombe genomic DNA library by complementation of cadmium hypersensitivity of a glutathione synthetase deficient mutant of S. pombe . A long open reading frame was found in the cloned DNA sequence. Amino acid sequence predicted from the long open reading frame coincided with amino acid sequences of peptides obtained by V8 protease digestion of the large subunit of the purified glutathione synthetase. The glutathione synthetase deficient mutant which harbored plasmids containing the glutathione synthetase large subunit gene exhibited glutathione synthetase activity higher than the activity in the wild type strain, though the plasmid did not contain the gene for the small subunit of the enzyme.

Localization of 350K molecular weight and related proteins in both the cytoskeleton and nuclear flecks that increase during G1 phase.

Monoclonal and polyclonal antibodies were raised against the highest molecular weight microtubule-associated protein (MAP-1) isolated from brain. Immunoblotting with the antibodies revealed the presence of cross-reactive protein of 350K or less on whole cells, isolated nuclei and cellular microtubules. Two-dimensional peptide maps showed substantial homology of immunoprecipitated cellular proteins of 350K, 80K and 51K with a 25K fragment of brain MAP-1. On antibody staining, immunofluorescence was seen on a cytoplasmic network, the mitotic spindle, the centrosome, and intranuclear flecks. The antibody causing immunofluorescence in all these sites was absorbed most effectively with slices of blotted membrane which contained the 350K protein. These results suggest that the cross-reactive molecules in diverse sites belong to the family of the 350K protein. The number of nuclear flecks and the amount of bound radioactivity of 125I-antibody almost doubled during G1 phase.

DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers

DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly alpha-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only beta-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase.

Conversion of DNA polymerase extracted from rat ascites hepatoma cells

Abstract DNA polymerase extracted fresh from rat ascites hepatoma cells possesses high molecular weight, maximal activity at neutral pH, and high sensitivity to N-ethylmaleimide (NEM). After physical and chemical treatment of the enzyme fraction, the appearance of low molecular weight DNA polymerase was detected by means of Sephadex gel filtration or sucrose density gradient centrifugation. This low molecular weight DNA polymerase possesses alkaline pH optimum, preference of native DNA as template/primer, and relative resistance to NEM.

DNA Chain Elongation Mechanism of DNA Polymerases α, β and γ

There are many lines of evidence that indicate the existence of at least two kinds of DNA replication mechanisms in eukaryotic cells. (1) One is that observed in nuclear DNA replication, where DNA chains (at least the lagging strand) are synthesized in relatively short pieces (3–5s) that are later elongated and joined together1–6. These short DNA intermediates are also observed in the replication of viral DNA such as polyoma virus7,8 and simian virus (SV)4O9,10. (2) The other is that for adenovirus DNA11-12 and mitochondrial DNA13. These DNA’s are not replicated via Okazaki pieces as the intermediates, but replicated in a continuous mode.

Isolation and characteristics of a leukemia-growth-promoting factor from calf thymus.

Leukemia-growth-promoting factor (LGPF) as we previously reported stimulates the growth of a murine leukemia subline (L17R) extensively. LGPF was isolated and 10(4) fold purified from calf thymuses by a combination of ammonium sulfate precipitation, Sephadex G-100 chromatography, hydroxylapatite chromatography, and Mono S-fast protein liquid chromatography (Mono S-FPLC). The mol. wt of LGPF was estimated to be approximately 25,000 a.m.u. from the elution pattern of Sephadex G-100 chromatography. The activity had high affinity for Mono S beads which are cation exchangers. Mono S fractions of LGPF are effective at a low concentration of 5 ng/ml. The activity was inactivated by heat (56 degrees C, 30 min), 1 mg/ml trypsin (37 degrees C, 1h), and 50 mM dithiothreitol (20 degrees C, 1h). The growth L17R leukemia cells are not only stimulated by LGPF, but also by pituitary and brain fibroblast growth factor (FGF). These data strongly suggest that LGPF is a heat sensitive cationic protein(s) acting as a member of FGF family.