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. Author manuscript; available in PMC: 2018 Dec 21.
Published in final edited form as: Mol Cell. 2017 Dec 7;68(6):1134–1146.e6. doi: 10.1016/j.molcel.2017.11.006

Figure 1. CDK4 phosphorylates p53-RS at Ser249 in G1/S phase and mediates its nuclear localization.

Figure 1.

A) The sequence comparison of p53WT and p53RS.

B) JNJ-7706621 (JNJ) inhibitor (Pan-CDK inhibitor) blocks p53-RS’s nuclear translocation. Cell fractions of flag-vector- or p53-RS-expressing HEK293 stable cell were treated by JNJ (2 μM or 10 μM) for 16 hours or U0126 (10 μM) for 4 h and then harvested for WB with indicated antibodies.

C) p53-RS interacts with CDK4 or CDK7. The same stable cells as above were synchronized with Nocodazole at 100ng/ml for 16 h and harvested at different time points (different phases) for IP with the anti-Flag antibody followed by WB with indicated antibodies.

D, E) CDK4 phosphorylates p53-RS in vitro. His-p53WT or His-p53RS was purified from E.coli for in vitro kinase reactions using Cyclin D1/CDK4 complexes commercially purchased. Phosphorylated proteins were detected by autoradiography or the anti-phosphor-S249 antibody.

F) p53-RS is phosphorylated at G1/S phase PLC/PRF/5 cells. PLC/PRF/5 cells were synchronized with Nocodazole, and harvested at G1/S phase for IP with IgG or p53 antibody followed by WB with indicated antibodies.

G) The majority of phosphorylated p53-RS in the nucleus. Synchronized PLC/PRF/5 cells were harvested at G1/S phase for subcellular fractionation followed by IP with IgG or the anti-p53 antibody followed by WB with indicated antibodies.

H) A CDK4 inhibitor PD033291 (PD) blocks the nuclear localization of endogenous p53-RS. PLC/PRF/5 cells were treated with or without PD (500 nM) for 16 h, and harvested for analysis of subcellular fractions of endogenous p53-RS by WB with indicated antibodies.