Fig. 5: Critical role of the N terminus of p15 in binding to and inhibiting CDK4 and CDK6.

a, b Domain switching between p15 and p16 narrows strong growth inhibitory activity of p15 to its N terminus. Mammalian expression vectors bearing Myc-tagged cDNA hybrids (i.e., p16-N terminus (p16-N) plus p15-C terminus (p15-C)) or p15-N terminus (p15-N) plus p16-C terminus (p16-C)), along with control vectors (Ctl) bearing Myc-tagged wild-type p15 or wild-type p16 were stably transfected and inducibly expressed in UMUC3 cells. pRb1 phosphorylation status 24 h after induced expression of wild type and hybrid proteins (a) and cell proliferation with time course indicated (b) were assessed by western blotting with semi-quantitation and WST-1 assay, respectively (a, right panels), were representative semi-quantitation by densitometry of the phosphorylated pRb1 shown in the left panel in reference to Myc-tag and β-actin. The experiments were repeated twice with similar results. b p16 and p16-N/p15-C were statistically compared as one group because of their highly similar readings and compared with p15 and p15-N/p16-C as individual groups. Note that p15-N/p16-C was as effective as wild-type p15 in inhibiting pRb1 phosphorylation and cell proliferation and, in comparison, that p16-N/p15-C was as ineffective as wild-type p16 in inhibiting pRb1 phosphorylation and cell proliferation. n = 5 of biologically independent samples. Data are presented as mean ± SD. Two-sided t-test was performed to compare the significance of the difference between the two groups (p15 versus p16; and p16-N/p15-C versus p15-N/p16-C). The P values are shown in the figure. c, d N-terminal deletion of p15 further narrows the critical region for CDK4 binding. Mammalian expression vectors bearing Myc-tagged deletion mutants of p15 cDNA as indicated above each panel, along with mock vector control (Ctl) and full-length (FL) p15 cDNA were stably transfected and inducibly expressed in UMUC3 cells. Immunoprecipitation (IP) followed by western blotting was then carried out (c). Alternatively, the histidine-tagged mutants were expressed in E. coli, immobilized on nickel column and incubated with total protein extracts from parental UMUC3 cell line (pull-down experiment, PD), followed by western blotting (d). Note that in both experiments the deletion mutant ∆2–15 reduced binding to CDK4 and the deletion mutants ∆2–27 and ∆3–49 completely abolished its binding ability to CDK4. Experiments in c and d were repeated three times with similar results.