Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

Abstract

Abstract. The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34 '~c2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wildtype FM3A cell line each possess at least three distinct histone HI kinases. HI kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34 r and the human cyclins A and B1. The results show that p34 r forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the nonpermissive temperature in vitro and in vivo. A third HI kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-1ike 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34 ~2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34 ~c~c~ is essential only during the G2-to-M transition in FT210 cells.

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

“…Recent evidence, however, has shown that cyclin A activates both the mitotic cdc2 kinase and the related cdk2 kinase that is required for transition of cells into S phase (Fang and Newport, 1991;Pagano et al, 1992). To establish beyond doubt that inhibition was mediated via the mitotic kinase, we took advantage of the recent identification of a murine cell-line (FT210) with a temperature-sensitive lesion in cdc2 kinase activity (Th'ng et al, 1990;Hamaguchi et al, 1992). Decline of cdc2, but not cdk2, activity occurs rapidly both in vivo and in vitro upon shifting from 32 to 39°C (Th'ng et al, 1990).…”

“…Immunoprecipitation of histone kinases from cyclin-activated extracts followed the methods described by Hamaguchi et al (1992). Cyclin-activated or control cytosol (200 Mig) in KEHM buffer (200 Ml) was incubated with anti-cyclin A or anti-cyclin B sera (1 MAl) for 2 h at 4°C.…”

Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis.

“…Entry into both S and M phase is controlled by cyclin-dependent kinases. Specialized cyclins and cdc2-1ike kinases control either the G1/S or the Gz/M transition in higher eukaryotes (Fang and Newport 1991;Hamaguchi et al 1992; Baldin et al 1993;Knoblich and Lehner 1993;Quelle et al 1993;Stem et al 1993; van den Heuwel et al. 1993;Knoblich et al 1994; for review, see Sherr 1993; Solomon 1993).…”

Repression of p27kip1 Synthesis by Platelet-Derived Growth Factor in BALB/c 3T3 Cells

“…Binding of growth-regulatory ligands results in the activation of signaling pathways that trigger direct alteration of specific metabolic pathways as well as immediate and delayed changes in gene expression (65). Mitogenic stimulation initiates a program of sequential synthesis of proteins, termed cyclins, that complex with and activate cyclin-dependent kinases (cdks) (9, 29-31, 39, 52, 54, 64, 67), enzymes that modulate key regulatory events leading to progression through and transitions between different stages of the cell cycle (7,13,20,21,47,55,64). The order of cyclin synthesis during cell cycle traverse has been examined in a variety of cells, including T cells, macrophages, epithelial cells, and fibroblasts (16,30,31,44,45,50,52).…”