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

Mita Gupta,Deborah Trott,Andrew C.G Porter

doi:10.1074/jbc.m607910200

Mita Gupta, Deborah Trott + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m607910200

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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.

Highlights

Transitions from G2 to mitosis and from G1 into S phase are key cell cycle control points
The results suggest a model in which two thresholds of Cdk1 activity must be exceeded sequentially during the cell cycle, the first to inhibit endoreduplication and the second to promote entry into mitosis
Human Cdk1 with T14A and Y15F substitutions (Cdk1-AF) Rescues HT2-19 Cells from Cdk1 Depletion— A plasmid carrying the human Cdk1 cDNA driven by the CDK1 promoter and the bacterial gpt gene, which confers resistance to mycophenolic acid and xanthine (MPA/ X), was previously shown to rescue HT2-19 cells from IPTG dependence [19]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Electroporation—HT1080 and HT2-19 cell culture and stable transfection by electroporation using a Gene Pulser electroporator (Bio-Rad) were as described [19]. PBSgptCDC was digested with NdeI and end-filled; the ϳ1-kb Cdk coding fragment was discarded, and the remaining 10.5-kb fragment was ligated to the 1-kb BamHI fragment of pUHD10-3-CDC2-HA or pUHD10-3-CDC2-A14F15-HA (kindly donated by David Morgan, University of California, San Francisco) carrying the Cdk coding sequence modified as described [23]. A CDK1 gene-targeting construct, pCDC/zeo, was made by inserting an end-filled 1.3-kb BamHI-SspI fragment from pZeoSV (Invitrogen) into pCDC2AX [19], linearized at an Asp718 site at the beginning of CDK1 exon 3. The following day, a mixture containing 600 nM siRNA and Oligofectamine (3% (v/v)) in Opti-MEM was prepared according to the manufacturer’s instructions and added to cells (100 or 500 ␮l/well) with 5 volumes of antibiotic-free medium, to give a final siRNA concentration of 100 nM.

Selection ratioc

RESULTS

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DISCUSSION