Rescue of a Human Cell Line from Endogenous Cdk1 Depletion by Cdk1 Lacking Inhibitory Phosphorylation Sites

Abstract

Cells that transiently overexpress cyclin-dependent kinase 1 lacking inhibitory phosphorylation sites (Cdk1-AF) undergo premature and catastrophic mitosis, reflecting the key role for Cdk1 in promoting a timely transit from G 2 into mitosis. Conversely, cells depleted of Cdk1 undergo repeated S phases without intervening mitoses (endoreduplication), reflecting a role for Cdk1 in preventing premature S phases. It is not known how Cdk1 prevents entry into S phase at times in G 2 when it does not promote mitosis. Also uncertain is the extent of redundancy between inhibitory phosphorylation and other mechanisms for controlling Cdk1 activity. We describe here human cells that not only tolerate stable Cdk1-AF expression but also rely on it for survival when endogenous Cdk1 is depleted. When residual endogenous Cdk1 expression is further depleted, however, proliferation of Cdk1-AF-rescued cells is inhibited. Interestingly, this inhibition is not accompanied by endoreduplication. These results are consistent with a two-threshold model for Cdk1 kinase activity, one for suppressing endoreduplication and one for promoting mitosis. They also indicate that inhibitory phosphorylation is indispensable for only a fraction of the total cellular complement of Cdk1. Cyclin-dependent kinases (Cdks)4 control eukaryotic cell cycle progression, ensuring that each phase occurs in an orderly and timely manner (1-3). To be active, Cdks must be bound to an appropriate cyclin molecule and free from inhibition by Cdk inhibitors. Their action can be further controlled by changes in subcellular localization and phosphorylation state. Cdks therefore have a variety of ways in which their activity can be modulated in response to extrinsic (e.g. presence of growth factor) or intrinsic (e.g. completion of DNA synthesis) cues (4 -8).Transitions from G 2 to mitosis and from G 1 into S phase are key cell cycle control points. Simple eukaryotes have a single Cdk (Cdk1, or Cdc2 and Cdc28 in budding and fission yeast, respectively) that controls both of these transitions. Higher eukaryotes have multiple Cdks (Cdk1 to Cdk11) with more specialized roles, but Cdk1 and its role in promoting mitosis have been conserved. In the normal cell cycle, mitotic cyclins (cyclins A, B, and B3) accumulate during G 2 , and Cdk1 is held in a relatively inactive state by inhibitory phosphorylation at residues Tyr 15 (in fission yeast) or Thr 14 and Tyr 15 (higher eukaryotes). Two kinases (Myt1 and Wee1) and one phosphatase (Cdc25) determine the phosphorylation status of these residues, and, together with Cdk1, these are thought to constitute a molecular switch that commits cells to undergo mitosis. Thus, in late G 2 , the sudden increase in Cdk1 activity that coincides with entry into mitosis is thought to be achieved by a double positive feedback loop in which Cdk1 kinase activity both inhibits Wee1 kinase and activates Cdc25 phosphatase (9). The resulting flood of Cdk1 activity promotes mitosis by phosphorylating a range of nuclear substrates (e.g. lamins and histone H3...

Preventing DNA over-replication: a Cdk perspective

“…Also consistent with the model in Fig. 2C is the ability of Cdk1 that lacks inhibitory phosphorylation sites, and is therefore unresponsive to Cdc25 phosphatase, to prevent endoreduplication in HT2-19 without promoting mitosis [ 37 ].…”

Ubiquitin-specific protease 22 is a deubiquitinase of CCNB1

“…Only weak USP22 phosphorylation was detected in HCT116 cells, presumably catalyzed by endogenous CDK1, and CDK1 overexpression significantly increased USP22 phosphorylation. In addition, expression of the constitutively active form of CDK1 (CDK1/AF) [ 37 , 38 ] further enhanced USP22 phosphorylation in HCT116 cells ( Figure 4b ). Moreover, mutation of the aspartic acid at position 146 of CDK1 (CDK1/D146N), which is a kinase-inactive mutant [ 39 ], completely abolished its ability to promote USP22 phosphorylation ( Figure 4b ).…”

Rapid Cycling and Precocious Termination of G1 Phase in Cells Expressing CDK1AF

“…However, several subsequent studies showed that although the introduction of CDK1AF does result in premature activation of CDK1 in HeLa cells, it has little or no effect on the timing of chromatin condensation and nuclear envelope breakdown (NEB) (Jin et al, 1996(Jin et al, , 1998Blasina et al, 1997). Moreover, one recent study has shown that human HT2-19 cells can be obtained that both tolerate and require CDK1AF for growth and division (Gupta et al, 2007). These results indicate that the inhibitory phosphorylation of CDK1, and the positive feedback loops that depend upon this inhibitory phosphorylation, are not essential for normal mitotic timing in HeLa cells and not essential for viability in HT2-19 cells.…”