Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

Abstract

Summary Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and non-physiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a non-catalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and non-inhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and non-redundant, rate-limiting roles in restriction point passage and S-phase entry.

Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways

“…We found that in the absence of 1NM-PP1, HCT116-CDK2 AS cells exhibit a modest proliferation defect compared with WT HCT116 cells (Fig. 3A), which is consistent with what has been previously reported (21). On treatment with 1NM-PP1, we found that proliferation of HCT116-CDK2 AS cells was further decreased, whereas we saw no effect in the HCT116-CDK2 WT cells (Fig.…”

“…In this report, we use a chemical genetic approach in which we replace endogenous WT CDK2 (CDK2 WT ) in transformed mouse embryonic fibroblasts (MEFs) and human colon cancer cells with an analog-sensitive (AS) version that is mutated to allow for acute and selective inhibition using modified ATP analogs. This approach has been used recently to identify a distinct, nonredundant role of CDK2 in cellular proliferation of nontransformed cells (21). Here, we show that small-molecule inhibition of CDK2 also disrupts cellular growth of transformed MEFs and human colon cancer cells, defining a role for CDK2 in cellular proliferation under the control of oncogenic signaling.…”

“…These cells were subsequently transformed by oncogenes c-MYC, HRASG12V, and v-ABL via retroviral transduction. HCT-116 CDK2 WT and CDK2 AS were generated by introducing human CDK2 AS alleles into the CDK2 locus using a recombinant adeno-associated virus (AAV) homologous recombination strategy that is described in detail elsewhere (21).…”

“…Importantly, HCT116 cells are amenable to homologous recombination for genetic ablation or replacement of candidate genes (27). We used engineered HCT116 cells, in which the CDK2 AS alleles were inserted into both endogenous CDK2 loci (HCT116-CDK2 AS ), replacing the endogenous WT gene by the method previously described for human CDK7 (21,28). We first asked whether acute inhibition of CDK2 could decrease proliferation of the HCT116 cells.…”

Cyclin-Dependent Kinases Regulate Ig Class Switching by Controlling Access of AID to the Switch Region

“…To uncover the possible role of CDK2 in regulating the nuclear level of AID, we took advantage of a cell line in which the wildtype CDK2 is replaced by an analog-sensitive molecule (CDK2AS) (25). CDK2AS is hypomorphic compared with the control RPEhTERT cells, and its activity can be restored or further inhibited by the adenine analogs 6-BAP or 3-MB-PP1, respectively.…”