Figure 4. CDK4/6 interactions and conformations.

(A) Illustration of regulatory CDK4/6 interactions and Rb activation. (B) Domain organization of CDK4, CDK6, and p16^INK4a; tested point mutations are listed. (C) 3D structure of CDK6 in complex with p16^INK4a. Crucial amino acids involved in the interaction of the two proteins are highlighted. The R31C mutant is depicted in orange (PDB code: 1BI7, Russo et al., 1998). (D) Protein:protein interaction (PPI) reporter analyses of the kinases CDK4 and CDK6 with p16^INK4a. Scheme illustrates CDK4/6 hetero-dimer formation with p16^INK4a analyzed using a PCA RLuc PPI reporter system. PPI induces the complementation of RLuc PCA fragments promoting an increase in bioluminescence (HEK293T cells, 48 hr of transient reporter expression). Bars represent the RLU fold change of PPI in relation to wild-type CDK4/6:p16^INK4a complex (mean ± SEM, n=7 ind. experiments). (E) Basal signal of CDK4/6 KinCon reporters with indicated mutations are shown (expressed for 48 hr in HEK293T cells) (mean ± SEM, n=6 ind. experiments). (F) Quantification of alterations of CDK4/6 KinCon reporter bioluminescence signals (HEK293T, expression for 48 hr) upon exposure to indicated CDK4/6i (1 μM) or DMSO for 3 hr (mean ± SEM, n=4 ind. experiments). Statistical significance for D–F: one-sample t-test (*p<0.05, **p<0.01, ***p<0.001).

Figure 4—figure supplement 1. Expression of CDK4/6:p16^INK4a PPI reporter constructs.

Exemplary western blot of CDK4/6 :p16^INK4a PPI PCA. Immunoblot with respective antibodies show expression levels of RLuc PCA F[1] and RLuc PCA F[2] tagged expression constructs.