During G1 phase of the division cycle, a eukaryotic cell commits to a round of duplication or enters a quiescent state (G0) (Morgan, 2007
). When conditions are appropriate for proliferation, growth factor signaling promotes passage through the restriction (R) point, after which further cell-cycle progression becomes growth factor-independent (Pardee, 1974
). This transition coincides with initiation of a transcription program directed by the E2F family of DNA-binding factors, which regulate genes critical for DNA synthesis (S) phase and mitosis (Bracken et al., 2004
). Analysis of gene expression in single cells suggests that the switch governing E2F activation is bistable in response to increasing growth factor concentration, attaining an ON or OFF steady state with no intermediate states of partial activation (Yao et al., 2008
G1/S transcription is controlled by both activator and repressor E2Fs that bind to a family of pocket proteins comprising the retinoblastoma tumor suppressor protein pRb, p107 and p130 (Cobrinik, 2005
). In G0 and early G1, gene expression is inhibited by repressor E2Fs, in complexes with p107 or p130 that bind E2F-responsive promoters and recruit histone-modifying enzymes to impose a repressive chromatin state (Frolov and Dyson, 2004
). Later in G1 and S phase, repression is relieved and free activator E2Fs, dissociated from pocket proteins, promote transcription. Cyclin-dependent kinases (CDKs) play a decisive role in this switch by phosphorylating pRb, p107 and p130. In vitro, cyclin D-, E- and A-dependent kinases phosphorylate pRb with overlapping but distinct site-specificity (Zarkowska and Mittnacht, 1997
), and multiple CDKs contribute to complete pRb inactivation in vivo (Lundberg and Weinberg, 1998
). Phosphorylations on distinct sets of residues—targets in vitro of different CDKs—decrease affinity between discrete regions of pRb and E2F (Rubin et al., 2005
; Burke et al., 2010
). The deregulation of pocket protein function and its control by CDKs is a hallmark of many cancers (Classon and Harlow, 2002
Cdk2 was originally implicated in this regulatory circuit because its period of cyclin-binding and activity extended from late G1 until just prior to mitosis (Pagano et al., 1992
; Rosenblatt et al., 1992
; Pagano et al., 1993
), and expression of dominant-negative Cdk2 arrested cells in G1, S and G2 phases (van den Heuvel and Harlow, 1993
; Hu et al., 2001
). This model was challenged by the discovery that Cdk2 is not essential for mouse viability (Berthet et al., 2003
; Ortega et al., 2003
). In cells lacking a full complement of catalytic subunits, Cdk1 can form complexes with cyclins D and E (Aleem et al., 2005
; Santamaria et al., 2007
); compensation by Cdk1 may explain how cells lacking Cdk2, Cdk4 and Cdk6 can proliferate in culture (Santamaria et al., 2007
). It remained unclear whether events early in the cell cycle depend exclusively on Cdk2 in wild-type cells, or if Cdk1 can perform these functions normally.
To begin to address this question, we previously determined the relative amounts of Cdk1 and Cdk2 bound to various cyclins during the course of a human cell cycle (Merrick et al., 2008
). Cyclins E and B bound almost exclusively to Cdk2 and Cdk1, respectively, whereas cyclin A formed complexes with both CDKs in strict temporal order—predominantly with Cdk2 until mid-S phase, and only thereafter with Cdk1. Cdk2 has priority despite being ~10-fold less abundant than Cdk1, possibly as a consequence of different activation mechanisms for the two kinases. In vivo, Cdk1 and Cdk2 follow distinct paths to full activity even though they are ~65% identical in sequence, have overlapping cyclin-binding profiles, and are targets of the same CDK-activating kinase (CAK)—the Cdk7 complex. The primary pathway for Cdk2 comprises two steps: first, phosphorylation of the activation (T-) loop by Cdk7 and then, binding to cyclin (Merrick et al., 2008
). Cdk1, conversely, cannot be phosphorylated by Cdk7 in the absence of a cyclin, but cannot form a stable complex without that phosphorylation, meaning that the two steps must occur in concert (Larochelle et al., 2007
). In vitro, a yeast CAK that phosphorylates monomeric Cdk1 can force it into the Cdk2 pathway, switching cyclin A-binding preference from Cdk2 to Cdk1 in extracts (Merrick et al., 2008
). This suggested that pairing rules for CDK-cyclin assembly derive from kinetic insulation of activation pathways.
Therefore an important function of Cdk2 may be to exclude Cdk1 from cyclin complexes until DNA replication has commenced, restricting Cdk1/cyclin A activity to later in S phase and potentially preventing precocious firing of late replication origins (Katsuno et al., 2009
). When that scaffolding function is preserved, moreover, Cdk2 activity might be required for early cell-cycle events, even though there is not a strict requirement for Cdk2 protein. Removing Cdk2 by gene disruption abolishes both catalytic activity and a non-catalytic function in restraining Cdk1 activation. To distinguish and define those roles, we needed an alternative strategy that would allow: 1) specific inhibition of Cdk2 catalytic activity; and 2) control over Cdk2-cyclin pairing and, consequently, Cdk1 complex assembly.
Here we replace wild-type Cdk2 in human epithelial cells with an analog-sensitive (AS) version modified to bind bulky adenine analogs. A Cdk2as allele is hypomorphic; the mutant protein has lost its competitive advantage over Cdk1 in binding cyclin A. That priority can be restored by different analogs, which either promote activation or inhibit activity of Cdk2as. We thus created a chemical “allelic series” that allows both rescue and selective antagonism of Cdk2 functions. Specific inhibition impedes cell proliferation, demonstrating an essential requirement for the catalytic activity of Cdk2. Both catalytic and scaffold functions are required for normal responses to growth factors and timing of R-point passage. Therefore, chemical genetics reveals Cdk2 to be a non-redundant, rate-limiting regulator of G1/S progression in human cells.