HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

Abstract

Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21^cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

ACKNOWLEDGMENTS

The early part of this work was supported by an NIH grant (1R01 CA76120) to the laboratory of William G. Kaelin, Jr., at the Dana-Farber Cancer Institute and Harvard Medical School, Boston. This work was supported by grants to P.D.A. from the W. W. Smith charitable trust and the V-foundation. Work in the laboratory of M.L. was supported by a grant from the Human Frontiers Science and the Association pour la Recherche sur le Cancer.

We thank Jianmin Gan for excellent technical assistance in generation of antibodies to HIRA. We thank Jon Chernoff and Randy Strich for critical reading of the manuscript and Wade Harper and Ken Zaret for discussion during the course of this work. P.D.A. thanks W.G.K. for his teaching and generous support.