HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2substrate whose expression blocks S-phase progression

Substrates of cyclin-edk2 kinases contain two distinct primary sequence mot ifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (cons ensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searc hed the database for proteins containing both of these motifs, One such pro tein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces, cerevisiae, Hir1p and Hir2p. Her e we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 k inase, 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 viv o on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of th ese, threonine 555, was phosphorylated by cyclinA-cdk2 in vitro. Third, pho sphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1) . Fourth, HIRA became phosphorylated on threonine 555 in S phase when cycli n-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic ex pression of HIRA in tells caused arrest in S phase and this is consistent w ith the notion that it is a cyclin-cdk2 substrate that has a role in contro l of the cell cycle.