Excessive cell proliferation induced by aberrant entry into the cell cycle is considered a hallmark of cancer. Commitment to cell cycle entry occurs during the G1 phase, when CDK4 and CDK6 form active complexes with one of the three D-type cyclins (D1, D2 or D3). These complexes promote G1-S transition in cancer cells by phosphorylating critical substrates, of which the Retinoblastoma tumor suppressor protein, RB1, as well as the related family members, RBL1 (p107) and RBL2 (p130), remain best characterized. Mechanistically, phosphorylation of RB proteins disables their function as transcriptional repressors to allow activation of the E2F-dependent transcriptional program, an important mediator of S-phase entry and initiation of DNA synthesis (Ortega et al., 2002
; Sherr and Roberts, 1999
). These processes are negatively regulated by INK4 proteins (including p15INK4
), which specifically inhibit the assembly and activation of cyclin D-CDK4/6 complexes.
It is therefore not surprising that CDK4 and its regulatory subunit, cyclin D1, are oncogenes; and recent findings have revealed that both are embedded in the ten most frequently amplified genomic loci in a diverse set of human cancers (Beroukhim et al., 2010
). Conversely, the gene encoding p16INK4
exhibits more deletions than any other recessive cancer gene (Bignell et al., 2010
). Moreover, cyclin D1-CDK4 is required for the formation of several tumor types, including breast and lung cancer, with the catalytic function of the CDK4 subunit being critically important (Yu et al., 2006
; Landis et al., 2006
; Puyol et al., 2010
Despite of this, the full spectrum of the substrates phosphorylated by CDK4/6 remains unknown, although this information is crucial for our understanding of kinase function in human cancer. It is also unclear whether individual cyclin D1/D2/D3-CDK4/6 complexes target the same subset of proteins for phosphorylation, or whether they possess distinct substrate specificities. Yet, linking CDK4/6 to their substrates is particularly challenging; unlike other CDKs, CDK4 and CDK6 are not readily susceptible to chemical genetics approaches, using ‘bulky’ ATP. Classical substrate-trapping methods also pose inherent limitations, such as the transient nature of physical kinase-substrate interactions, the general difficulty to detect low-abundance proteins, and the experimental restriction of the analysis to certain cell or tissue types.
Here we sought to overcome these limitations, and to uncover genuine substrates of CDK4/6 across the human proteome. Through functional analysis of substrate phosphorylation we aim to define mechanisms by which CDK4/6 promote tumorigenesis in order to maximize the merits of CDK4/6 small molecule inhibitors for targeted therapy.