The entry into mitosis is controlled by CDK1/Cyclin B, also known as MPF (Maturation Promoting Factor). Previous research has shown that cyclin B synthesis is the key for driving the embryonic cell cycle and determining the timing of mitosis in
Xenopus leavis [
1,
2,
3]. Apparently, it is a key, but not the only controlling factor. Cyclin B accumulation during interphase is necessary for CDK1 activation because without cyclin CDK1 cannot be active as a protein kinase. On the other hand, cyclin degradation through the ubiquitin pathway is necessary for CDK1 inactivation and mitotic exit [
1,
2,
3]. Cyclin B is
encoded by
several genes and creates a family of B-type cyclins required for mitosis (e.g.,
cyclins B1, B2, and B3
in human; or cyclins B1-B5 in
Xenopus leavis [
4,
5]).
De novo B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation [
4]. However, despite a steady increase in cyclin B levels in G2 [
6], CDK1 activation is biphasic characterized by a slow phase, followed by a rapid phase attributed to the positive feedback between newly activated CDK1 and its major activating phosphatase CDC25 [
6]. It was intriguing for the long time how the stable increase in cyclin B level may lead to this biphasic CDK1 activation before the CDK1/CDC25 positive feedback acceleration triggering the final pic of CDK1 activity followed by its inactivation linked with cyclin B degradation (ibid.). This particularity suggested that the control of CDK1 activation at the initial stages of its mitotic activation might not solely depend on cyclin B accumulation, but might also involve an unidentified inhibitor that could counterbalance CDK1 activation assuring slow and biphasic mode of this process. Our research group performed a proteomic screen to find novel CDK1 partners in M-phase-arrested
Xenopus leavis eggs
vs. freshly activated ones (5 minutes post activation; [
7]). We showed a rapid, taking 5 min., change in the composition of the CDK1 complex during the period between the MII arrest of oocytes and their activation for development. However, we did not find in this screen any classical CDK1 inhibitors, like INK4 or Cip/Kip, which potentially could slow down CDK1 activation [
8] in a CDK1 complex. Surprisingly for us, we found a CDC6 protein associated with the active mitotic CDK1. CDC6 is an evolutionarily conserved member of the AAA + ATPase family well known as the S-phase activator [
6]. Our further results showed that a CDC6-dependent mechanism inhibits CDK1 throughout the whole period of the M-phase (including the highest pic of CDK1 activity just prior its inactivation). Moreover, CDC6 associated with CDK1 during the M-phase rapidly dissociates from this kinase immediately after CDK1 inactivation suggesting that once CDK1 fully inactivated by separation from cyclin B, the association with CDC6 becomes unnecessary. It was a surprising observation in the light of the knowledge at that time suggesting a potential role of CDC6 only in the mitotic exit, and not entry and progression [
9]. Therefore, we became particularly intrigued by the role of CDC6 in the M-phase regulation and especially in the control of the dynamics of CDK1 mitotic activation and pursuit this research. The important hint to CDC6 role in CDK1 activation process was its mentioned above function in the M-phase exit described in
yeast and human cells [
10,
11,
12]. Through biochemical analysis of Xenopus embryo cell-free extracts we showed that a recombinant CDC6 protein acts as an inhibitor of CDK1 during the first embryonic mitosis in
Xenopus leavis [
9,
13,
14,
15,
16,
17]. Importantly, when endogenous CDC6 is depleted, the first, initial and slow phase of CDK1 activation is removed, and the kinase activation ceases to be biphasic. This, in turn, accelerates the timing of mitosis and the pic of CDK1 activity induced by the CDC25/CDK1 activation loop [
9,
13,
14,
15,
16,
17]. Moreover, we confirmed that it is also the case in the mouse embryos, which suggested a universal inhibitory role of CDC6 in CDK1 activation (ibid.). Furthermore, we showed that two proteins: CDC6 and Xic1 (the last one is a
bona fide CDK1 inhibitor identified for its role in later stages of
Xenopus embryo development) interact within the mitotic CDK1 protein complex. Upon arriving at the peak of the CDK1 activity (measured by histone H1 kinase activity), the Xic1 momentarily dissociates from the mitotic CDK1 allowing maximum activation of CDK1 during the M-phase [
9]. The identification of this new inhibitory mechanism towards CDK1 was surprising because it has been well known already for years that CDK1 is inhibited by the posttranslational modification by Wee1/Myt1 kinases. Thus, our data showed that there are two independent mechanisms of CDK1 activity in the game during the mitotic entry.
For this reason, we provide here an overview of the molecular processes underlying timely mitotic entry and progression. We focus on the CDK1 regulatory network and especially on the function of CDC6/Xic1 and Wee1/Myt1 acting as CDK1 inhibitors. The multiplied system of CDK1 activators and inhibitors explains how the mitotic activation of CDK1 is tuned and why cells divide in the strictly defined time frame. We also explain how the various pathways regulating this process are integrated.