p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity

Abstract

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF. Although primarily proposed to require a functional p53·Mdm-2 signaling axis, recently p14ARF has been implicated in p53-independent cell cycle regulation. Here we show that p14ARF preferentially induces a G 2 arrest in tumor cells lacking functional p53 and/or p21. Expression of p14ARF impaired mitotic entry and enforced a primarily cytoplasmic localization of p34cdc2 that was associated with a decrease in p34cdc2 kinase activity and reduced p34cdc2 protein expression. A direct physical interaction between p14ARF and p34cdc2 was, nevertheless, ruled out by lack of co- immunoprecipitation. The p14ARF-induced depletion of p34cdc2 was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G2-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34cdc2 using a constitutively active, phosphorylation-deficient p34cdc2AF mutant alleviated this p14ARF-induced G2 arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14 ARF arrests cells lacking functional p53/p21 in the G2 phase of the cell cycle by targeting p34cdc2 kinase. This may represent an important fail-safe mechanism by which p14ARF protects p53/p21-deficient cells from unrestrained proliferation. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.