Abstract
Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK–activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21cip1. In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.
Highlights
The cell cycle commitment at the restriction (R) point in G1 phase is initiated by inactivating phosphorylations of the central cell cycle/tumor suppressor pRb by CDK4 and CDK6, which are activated by D-type cyclins induced by mitogenic/oncogenic signaling [1,2,3]. pRb phosphorylation is maintained independently of cyclins D, and of mitogens, by a positive feedback loop linking pRb to E2F-dependent transcription of cyclin E, which leads to CDK2 activation and further phosphorylation of pRb [4]
The phosphorylation of cyclin D1-bound CDK4 appeared at 2–3 h into G1 phase, whereas the phosphorylation of cyclin D3-bound CDK4 was detectable in serum-deprived cells and increased much later at 12 h and subsequent time points, when most cells were in S–G2 phases (Figure S1C)
To test whether CDK7 inhibition affects the activation of CDK4 through T172-phosphorylation, serum-deprived wild-type and K7AS HCT116 cells were re-stimulated by serum in the continuous presence or absence of the bulky adenine analog 1-NMPP1 (10 mM) to inhibit CDK7 activity
Summary
The cell cycle commitment at the restriction (R) point in G1 phase is initiated by inactivating phosphorylations of the central cell cycle/tumor suppressor pRb by CDK4 and CDK6, which are activated by D-type cyclins induced by mitogenic/oncogenic signaling [1,2,3]. pRb phosphorylation is maintained independently of cyclins D, and of mitogens, by a positive feedback loop linking pRb to E2F-dependent transcription of cyclin E, which leads to CDK2 activation and further phosphorylation of pRb [4]. The cell cycle commitment at the restriction (R) point in G1 phase is initiated by inactivating phosphorylations of the central cell cycle/tumor suppressor pRb by CDK4 and CDK6, which are activated by D-type cyclins induced by mitogenic/oncogenic signaling [1,2,3]. PRb phosphorylation is maintained independently of cyclins D, and of mitogens, by a positive feedback loop linking pRb to E2F-dependent transcription of cyclin E, which leads to CDK2 activation and further phosphorylation of pRb [4]. This, together with other positive-feedback circuits, such as E2F inducing its own transcription and the mutual inhibition between cyclin E-CDK2 and p27Kip, has been shown to generate a bistable pRb-E2F switch to convert graded mitogen inputs into all-or-none E2F responses and cell cycle commitment [5]. P21 is the main transcriptional target involved in replicative senescence and p53-dependent cell cycle inhibition in response to DNA damage.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
