|
Comment |
A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Fisher RP et al. Phosphorylation by the CDK-activating kinase (CAK) is a required step in the activation of cyclin-dependent kinases. We have purified CAK from mammalian cells; the enzyme comprises two major polypeptides of 42 and 37 kDa. Protein sequencing indicates that the 42 kDa subunit is the mammalian homolog of MO15, a protein kinase known to be a component of CAK in amphibians and echinoderms. Cloning of a cDNA encoding the 37 kDa subunit identifies it as a novel cyclin (cyclin H). We have reconstituted CAK in vitro with the MO15 catalytic subunit and cyclin H, demonstrating that MO15 is a cyclin-dependent kinase (CDK7). Like other CDKs, MO15/CDK7 contains a conserved threonine required for full activity; mutation of this residue severely reduces CAK activity. The CAK holoenzyme activates complexes of CDK2 and CDC2 with various cyclins and also phosphorylates CDK2, but not CDC2, in the absence of cyclin. Thus, CAK is a CDK-cyclin complex implicated in the control of multiple cell cycle transitions.
CDK7 and CCNH Are Components of CDK-Activating Kinase and Are Required for Meiotic Progression of Pig Oocytes. Fujii W et al. CDK-activating kinase (CAK) phosphorylates threonine 161 (T161) of CDC2, a catalytic subunit of maturation/M-phase promoting factor (MPF), and is essential for MPF activation in mitosis. CAK has been thought to consist of a catalytic subunit, a regulatory subunit and an assembly factor: CDK7, CCNH (also known as Cyclin H) and MNAT1 (also known as MAT1), respectively. Although it is known that the meiotic progression of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in the meiotic progression of porcine immature oocytes. The T161 phosphorylation of CDC2 was found around germinal vesicle breakdown (GVBD) and thereafter, from 18 h to 48 h of culture. The GVBD rate at 18 h was increased by the overexpression of CDC2, but not mutated CDC2 (T161 replaced by alanine). Transcripts of CDK7, CCNH and MNAT1 were detectable throughout the culture period and their protein distribution patterns during oocyte maturation were the same as those reported in mitotic somatic cells. Overexpression of CDK7 or CCNH accelerated the meiotic events, such as meiotic resumption, T161 phosphorylation of CDC2, CCNB (also known as Cyclin B) synthesis and MPF activation. On the contrary, knockdown of CDK7 or CCNH caused the inhibition of these meiotic events. In contrast, overexpression and antisense RNA injection of MNAT1 had no influence on meiotic resumption, the status of T161 phosphorylation of CDC2, or MPF activity. These results suggest that CDK7 and CCNH activate CDC2 by T161 phosphorylation, and comprise CAK, which is required for normal meiotic progression during porcine oocyte maturation.
|