Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).

Abstract

Abstract. The activity of cyclin-dependent kinases (cdl~) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34 ~c2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating _ki-nase), and it was shown to contain p40 M°15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40 M°15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40 M°tS, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coll. These tools were then used to demonstrate that p40 M°~5 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40 u°15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40 u°15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34 ~c2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

Nature of the Critical Labile Event That Controls RB Phosphorylation in the Cyclic AMP-Dependent Cell Cycle of Thyrocytes in Primary Culture

“…The potential regulation of CDK4 activity by phosphorylation is not easily studied since it is not associated with modifications of electrophoretic migration. CAK is generally considered to be constitutively active during cell cycle [37], but this should be demonstrated in our system. Very recently, the inhibitory tyrosine phosphorylation of CDK4 has been reported to be involved not only in DNA damage-induced G1 arrest, but also in regular cell's arrest in quiescence [38].…”

Interferon-induces expression of cyclin-dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK)

“…Since we were not able to completely restore the CDK2 H-1 kinase activity by immunodepletion of CKI, we examined the possibility whether CDK-7 could be a potential IFN target using a well-characterized anti-CDK-7 mAb (Tassan et al, 1994). Figure 5 shows that IFN-a had no inhibitory e ect on CDK-7 expression except that we noticed the appearance of a fused band (not resolved fully in this gel, shown by an arrow) of a slightly faster moving band at 24 h post IFN-a-treatment (Figure 5a, lane 5).…”

Ubiquitin/proteasome-mediated degradation of p19INK4d determines its periodic expression during the cell cycle

“…(a) Skin diploid ®broblasts (strain AG01518B) were synchronized by contact inhibition essentially as described in Figure 1(a). After replating to lower density, the cell cycle re-entry was monitored by¯ow cytometry, and at the indicated cell cycle phases, the abundance of p19 INK4d was analysed by immunoblotting, and compared with a stable and non-oscillating protein CDK7 (Tassan et al, 1994), used as a loading control. Where indicated, the cells were treated with LLnL (25 mM) for 4 h before lysis.…”