Cip/Kip cyclin-dependent protein kinase inhibitors and the road to polyploidy

Abstract

Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development.

Figure 1

Developmentally regulated polyploidy. Normal mitotic cell cycles results in two diploid mononucleated daughter cells with each nucleus containing two copies of each homologous chromatid (2N). Re-replication of DNA during S-phase is an aberrant event that produces giant nuclei and apoptosis. However, developmental signals can induce cells to become polyploid either by completing mitosis in the absence of cytokinesis [(C^-), acytokinetic mitosis], or by melding two G0-phase cells into a single cell containing two G-phase nuclei (cell fusion), or by arresting cells in G2-phase and then inducing another S-phase (endoreduplication), or by arresting cells in M-phase (M*) in the absence of cytokinesis (endomitosis). Multiple rounds of acytokinetic mitosis produce multinucleated giant cells. Multiple cell fusion events produce multinucleated myotubes in skeletal muscle. Multiple rounds of endoreduplication (endocycles) produce mononucleated giant cells, whereas multiple rounds of endomitosis produce a single multilobular nucleus.

Figure 2

The role of p27^Kip1 in preventing premature activation of S-phase. Cdk1:CcnA and Cdk1:CcnB activities are both required to induce mitosis during mammalian cell proliferation. Either activity is required to activate the APC^Cdc20 ubiquitin ligase which, in turn, targets the mitotic cyclins A and B for ubiquitin mediated degradation by the 26S proteasome. Cdk1:CcnA also phosphorylates the Orc1 subunit and thereby prevents ORC binding to chromatin until metaphase is completed and APC^Cdc20 inhibits Cdk1 activity by reducing the pools of mitotic cyclins A and B. Once this occurs, and the spindle checkpoint releases APC^Cdc20, cells move into anaphase and APC^Cdc20 targets geminin for degradation. In the absence of CDK activity, Orc1 and Cdc6 are dephosphorylated and bind to chromatin. In the absence of geminin, Cdt1 loads the MCM DNA helicase onto ORC•Cdc6•chromatin sites. Cdk1 protein is inactivated by the Wee1 and Myt1 protein kinases. APC^Cdh1 appears and targets Cdc20 for ubiquitin-dependent degradation. The cells are now maintained in G1-phase by APC^Cdh1 preventing the synthesis of cyclins A and B. However, cyclin E is not an APC substrate. As cyclin E appears, Cdk2:CcnE activity activates the replication complexes and drives the cells into S-phase. The role of p27 is to prevent premature accumulation of Cdk2:CcnE activity.

Figure 3

Fluorescence activated cell sorting (FACS) analysis of mouse trophoblast stem (TS) cells undergoing mitotic cell cycles or endoreduplication as they differentiate into trophoblast giant (TG) cells when deprived of FGF4. p57-/- TS cells respond to the same conditions by forming multinucleated TG cells. Data are from [18].

Figure 4

The role of p57^Kip2 in the initiation and maintenance of endocycles. (A) Endocycles are initiated in mouse TS cells in the absence of FGF4. When p57 is expressed in TS cells as a consequence of FGF4 deprivation, Cdk1 activity is inhibited. Consequently, APC^Cdh1 will be assembled instead of APC^Cdc20. Orc1 will not be phosphorylated. In the absence of phosphorylation, any Orc1-P and Cdc6-P produced during S and G2-phases by Cdk2:CcnA will be dephosphorylated by cellular protein phosphatases. In the absence of CDK activity, APC^Cdh1 is assembled and targets geminin for degradation. Thus, the cell has effectively entered a G1-phase-like state without passing through mitosis. (B) Based on studies of endocycles in Drosophila follicle cells, Cdk2:cyclin E activity oscillates such that it is high during the S-phases and low during the G-phases. In contrast, APC^Cdh1/Fzr activity is high during G-phases and low during S-phases [40,41]. This is in keeping with the fact that Cyclin E:Cdk2 inactivates Cdh1/Fzr [51] suggesting that APC^Cdh1/Fzr oscillations are driven by periodic inhibition of Fzr by Cyclin E:Cdk2 [40,41]. Similarly, geminin levels are high during S-phase and low during gap phase [41]. In cells programmed for endoreduplication, inhibition of Cdk1 activity results in premature assembly of APC^Cdh1/Fzr. Thus, a feedback loop exists that reinforces inhibition of Cdk1 activity triggered by endocycle entry. In the absence of Geminin and CDK-dependent protein phosphorylation, preRC assembly occurs as though cells had entered G1-phase. Symbols: ⊥ indicates target is inhibited, +→ indicates target is activated, → indicates product of reaction.

Figure 5

Role of p21^Cip1 in preventing apoptosis during endocycles. Upon completion of DNA replication in mitotic cell cycles, Cdc25 protein phosphatase and cyclin B are transported into the nucleus where they convert the inactive form of Cdk1 into an active form that can bind to cyclins and initiate mitosis. ATR is a protein kinase that senses the presence of excess single-stranded DNA resulting either from stalled replication forks, or from DNA damage. ATR then phosphorylates Chk1. This activates the Chk1 protein kinase and it, in turn, phosphorylates Cdc25, inhibiting it and thereby preventing cells from entering mitosis until the problem is corrected. p21 either directly or indirectly prevents expression of Chk1 protein. Under these conditions, cells do not trigger apoptosis in response to incomplete DNA synthesis or DNA damage.