Abstract
M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca++/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.
Highlights
Rapid M-phase Promoting Factor (MPF) activation is granted by an activation loop in which the cdc25C phosphatase removes inhibitory phosphorylations of cdk1 at thr-14 and tyr-15, while MPF stimulates cdc25C activity and lowers activity of wee1, the cdk1 tyr-15 kinase [1,2]
By analying samples taken at short intervals (2 min) across the mitosis-to-interphase transition we repeatedly found that total egg extract’s histone H1 kinase [10], declined ahead of the cyclin B1 content and concomitantly with the decline of cyclin A (Fig. 1 A; upper panels; cyclins were detected from total extracts aliquots by immunoblot and total histone H1 kinase by autoradiograph of phosphorylated histone; P-HH1; note the decline in activity between 32 to 34 min and the stability of cyclin B1)
Our data show that timely completion of M-phase requires proteolysis-independent control of MPF activity
Summary
Rapid MPF activation is granted by an activation loop in which the cdc25C phosphatase removes inhibitory phosphorylations of cdk at thr-14 and tyr-15, while MPF stimulates cdc25C activity and lowers activity of wee, the cdk tyr-15 kinase [1,2]. At Mphase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis inactivates (Peters 2002). Cyclin degradation is initiated by activation of the ubiquitin ligase APC/C associated with its coactivator Cdc (APC/CCdc20) [3]. Several APC/C subunits and Cdc undergo MPF-dependent phosphorylation during M-phase [3]. Phosphorylation of APC/C stimulates its ubiquitin-ligase activity, phosphorylation of Cdc hampers binding to APC/C [3,4,5]. MPF activity may play both positive and negative actions on APC/CCdc activation [6,7]. Evidence suggests that interruption of the MPF activation loop may play a role for timing M-phase exit [8,9]. No direct evidence that the MPF activation loop is interrupted at M-phase exit has been provided. We set out to gain insight into this matter using the in vitro cell cycle system derived from Xenopus eggs
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