Hideyo Yasuda

Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human cdc25B phosphatase.

In mammalian cells, p34 cdc2 kinase undergoes phosphorylation at threonine‐14, tyrosine‐15 and threonine‐161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine‐14 and tyrosine‐15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine‐15 in vitro, but whether it also does at threonine‐14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine‐14 and tyrosine‐15 but not threonine‐161.

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.

Microtubule destabilization by cdc2/H1 histone kinase: Phosphorylation of a “Pro-rich region” in the microtubule-binding domain of MAP-4

Microtubule-associated protein-4 (MAP-4), a major MAP in proliferating cells, consists of a microtubule-binding domain and a projection domain protruding from the microtubule wall. The former contains a Pro-rich region and an assembly-promoting (AP) sequence region which is common to the neuron-specific MAPs, MAP-2 and tau1. In this paper, we describe the phosphorylation of the Pro-rich region of MAP-4 and the suppression of its assembly-promoting activity by cdc2/H1 histone kinase. This inactivation of MAP-4 may cause disassembly of the interphase microtubular network at the end of the G2 phase of the cell cycle.

The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase

The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.

THE DIFFERENCE IN MURINE CDC2 KINASE ACTIVITY BETWEEN CYTOPLASMIC AND NUCLEAR FRACTIONS DURING THE CELL CYCLE

The mouse analog of yeast CDC2+ kinase was detected in the cytoplasmic and nuclear fractions of cultured mouse FM3A cells. Its activity in the nuclear fraction increased in the G2/M phase became seven times higher than that in the G1/S phase, while the activity in the cytoplasmic fraction remained was almost constant from the G1/S to G1 phases. The activity in the cytoplasmic fraction was similar to that in the nuclear fraction in the G2/M phase. The amount of the enzyme remained almost constant during the cell cycle in both the nuclear and cytoplasmic fractions. These findings suggest that the cytoplasmic enzyme might play an independent role in the cell cycle.

A temperature-sensitive CHO-K1 cell mutant (tsTM13) defective in chromosome decondensation and spindle deconstruction in M phase

A temperature-sensitive CHO-K1 cell mutant, tsTM13, exhibited a delayed cell cycle progression from metaphase to telophase at a nonpermissive temperature and was finally arrested from anaphase to telophase. Metaphase chromosomes were overcondensed and chromosome disjunction in anaphase was uncoordinated. In telophase, sister chromatids were segregated and cytokinesis was completed, but chromosome structure remained in a condensed state and the spindle was not deconstructed. The level of phosphorylation of histones H1 and H3 remained high in the later stages of mitosis and the activity of histone H1 kinase was also maintained at a high level. These results strongly suggest that the pleiotropic defects of tsTM13 cells in mitosis are associated with a lack of inactivation of activated histone H1 kinase.

Cyclin-dependent kinase 2 (cdk2) in the murine Cdc2 kinase TS mutant.

Kinases of the mammalian cdc2 family including cdk2 (cyclin-dependent kinase 2) are thought to be involved in both the G2/M transition and DNA replication. To investigate the role of cdc2 kinase and cdk2 in cell cycle progression, murine tsFT210 cells bearing a temperaturesensitive cdc2 mutation were used. These kinases were purified by column chromatography, using a peptide with the consensus phosphorylation site of cdc2 kinase as the substrate. In this mutant, cdc2 kinase activity was temperature sensitive and cdk2 activity was not. At the restrictive temperature, the mutant was only arrested in the G2 phase and not in the G2-S phase, suggesting that cdk2 did not compensate for cdc2 kinase at the G2/M transition but did function at the G1-S phase. This suggestion was supported by the finding that transfection of cdk2 cDNA did not improve the growth of the mutant cell line at the restrictive temperature, although transfection of cdc2 cDNA did.