Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.

In response to DNA damage, p53 induces either cell-cycle arrest or apoptosis by differential transcription of several target genes and through transcription-independent apoptotic functions. p53 phosphorylation at Ser46 by HIPK2 is one determinant of the outcome because it takes place only upon severe, nonrepairable DNA damage that irreversibly drives cells to apoptosis. Here, we show that p53 represses its proapoptotic activator HIPK2 via MDM2-mediated degradation, whereas a degradation-resistant HIPK2 mutant has increased apoptotic activity. Upon cytostatic, nonsevere DNA damage, inhibition of HIPK2 degradation is sufficient to induce p53Ser46 phosphorylation and apoptosis, converting growth-arresting stimuli to apoptotic ones. These findings establish HIPK2 as an MDM2 target and support a model in which, upon nonsevere DNA damage, p53 represses its own phosphorylation at Ser46 due to HIPK2 degradation, supporting the notion that the cell-cycle-arresting functions of p53 include active inhibition of the apoptotic ones.

During normal cell cycles, the function of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, is required for G 2 phase progression. Accordingly, the G 2 arrest induced by DNA damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the G 2 arrest induced by DNA damage. We asked whether the CCAATbinding NF-Y modulates mitotic cyclins, cdk1, and cdc25C gene transcription during this type of G 2 arrest. In our experimental conditions, the integrity of the CCAAT boxes of cyclin B1, cyclin B2, and cdc25C promoters, as well as the presence of a functional NF-Y complex, is strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominantnegative p53 protein, impairing doxorubicin-induced G 2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during p53-dependent G 2 arrest induced by DNA damage. These data suggest a transcriptional regulatory role of NF-Y in the G 2 checkpoint after DNA damage.