A Monoclonal Antibody Against the PSTAIR Sequence of p34^cdc2, Catalytic Subunit of Maturation‐Promoting Factor and Key Regulator of the Cell Cycle

Abstract

A homolog of the serinehhreonine protein kinase (~3 4~~~' ) , encoded by the cdcZf gene of the fission yeast (Schizosaccharomyces pombe), is a catalytic subunit of maturation-promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of ~3 4~~~' This antibody recognizes 31 -34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either ~3 4~~~' itself or proteins highly homologous to ~3 4~~~' in the given species, since, in all species studies to date, they are all precipitated with p13suc1, the fission yeast S U C~+ gene product, which binds to ~3 4~~~' with high specificity. The anti-PSTAIR imrnunoprecipitate had no histone H1 kinase activity and did not contain cyclin 6, suggesting that the PSTAIR region is masked when ~34'~'' forms a complex with cyclin B as an active kinase. lmmunoblotting with the anti-PSTAIR antibody demonstrated that the fastest-migrating form of ~3 4~~~' homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34cdc2 The observed broad cross-reactivity of the anti-PSTAIR antibody against ~34'~'' homologues in various species should permit us to examine the role of ~34'~'' homologues in the regulation of the cell cycle in a variety of organisms.

A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

“…In animals, PSTAIRE CDKs include three types, i.e. cdc2/CDK1, CDK2 and CDK3 [20][21][22]. In plants, PSTAIRE CDKs have been identified in several species, such as Arabidopsis (CDC2a), maize (cdc2ZmA), rice (cdc2Os-1 and cdc2Os-2), alfalfa (cdc2MsA and cdc2-MsB) and Fucus [9,10].…”

Possible Involvement of Phosphatidylinositol 3-Kinase, but Not Protein Kinase B or Glycogen Synthase Kinase 3β, in Progesterone-Induced Oocyte Maturation in the Japanese Brown Frog, Rana japonica

“…The bacterially produced protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution in Tris-glycine buffer without SDS, according to the method described previously (Hirai et al, 1992). The purified protein was dialyzed against 1 mM HEPES (pH 7.0), lyophilized, and injected into two mice to produce antibodies as described previously (Yamashita et al, 1991). Polyclonal antibodies against the recombinant PKB were affinity-purified with the anti-genic proteins electroblotted onto an Immobilon membrane (Millipore, Tokyo, Japan).…”

“…The blots were probed with the following antibodies: anti-Xenopus PKB mouse polyclonal antibody cross-reactive with Rana PKB (this study), anti-Xenopus GSK3β mouse monoclonal antibody crossreactive with Rana GSK3β (Santa Cruz Biotechnology), and antiMyc mouse monoclonal antibody (9E10). The antigen-antibody complex was visualized by alkaline phosphatase-conjugated secondary antibodies, as previously described (Yamashita et al, 1991).…”

Somatic Lamins in Germinal Vesicles of Goldfish (Carassius auratus) Vitellogenic Oocytes.

“…Separated proteins were electrophoretically blotted onto the PVDF membranes (Millipore, Bedford, MA, USA). Western blot analysis was done by methods previously reported (Yamashita et al, 1991). Monoclonal antibodies (L200 and O3E6, see below) were used as primary antibodies.…”

“…The second and third incomplete adjuvant injections were with or without adjuvant, respectively. Fusion and screening methods were according to Yamashita et al (1991). Initial screenings were performed by ELISA plates, Xenobind (Denopore Corp., Hawthorne, NJ, USA) conjugated with electroeluted rainbow trout lamins (10 ng/ml) from SDS-PAGE gels.…”