Homeodomain-Interacting Protein Kinase 1 Modulates Daxx Localization, Phosphorylation, and Transcriptional Activity

Homeodomain-interacting protein kinase 1 (HIPK1) is one of three closely related serine/threonine protein kinases that regulate the activity of a broad range of transcription factors (5, 10, 15, 22, 23). The HIPKs were originally identified as nuclear protein kinases that function as corepressors for various homeodomain-containing transcription factors. Subsequently, HIPK2 was shown to provide a regulatory role for a large transcriptional repressor complex, as it interacts with the repressive elements Groucho and histone deacetylase (HDAC1) (4). HIPK2 also associates with high-mobility group I (y) proteins, a family of nuclear architectural proteins that influence transcription regulation (26). The HIPKs are related to a group of kinases that includes Yak1 (Saccharomyces cerevisiae), YakA (Dictyostelium discoideum), and Minibrain (Drosophila melanogaster, rodents, and humans) (15, 23). This group represents a family of protein kinases that regulate the transition from growth to differentiation in eukaryotic cells. The assignment of HIPKs to this group suggests a common function in growth regulation. In fact, growth-regulatory and tumor suppressor functions have been attributed to the HIPKs (5, 10, 26, 28). For example, HIPK2 transfection into cells results in a dramatic decrease in colony formation (5, 10). This outcome occurs through the cooperative activities of HIPK2 and the tumor suppressor protein p53 (5, 10). HIPK2 phosphorylates p53 on Ser 46, resulting in activation of p53-dependent transcription, cell growth regulation, and apoptosis initiation (5, 10, 31). A potential apoptotic function was also assigned to HIPK3, as it interacts with the cell surface death receptor Fas and phosphorylates the Fas-associated death domain (FADD), a transducer of Fas-mediated apoptotic signaling (28). The association of HIPK3, a nuclear protein, with a cell surface receptor was reconciled by demonstrating diffuse cytoplasmic HIPK3 localization. Daxx is also a nuclear protein that was shown to associate with cytoplasmic and cell surface molecules, including transforming growth factor beta and Fas (25, 32). Overexpressed Daxx was shown to bind the Fas death domain and to mediate apoptotic signaling by activating ASK-1 and subsequently the Jun-N-terminal kinase (JNK) pathway, independent of the FADD/procaspase-8 pathway (3, 32). Within the nucleus, Daxx interacts with the promyelocytic leukemia protein (PML) and localizes to PML oncogenic domains (PODs) (17, 30, 33). Localization of Daxx to PODs correlates with Daxx's proapoptotic activity. For example, Daxx mutants that fail to localize to PODs do not facilitate Fas-induced cell death (30). Furthermore, in the absence of PML, Daxx is dispersed throughout the nucleus, and activated cell death is diminished (33). These findings indicate that Daxx and PML may cooperate in PODs in mediating apoptotic signals. This may be important in understanding the biological nature of acute promyelocytic leukemia, which is characterized by reciprocal chromosomal translocation between the pml and retinoic acid receptor α genes, resulting in the oncogenic fusion product PML-retinoic acid receptor α. In acute promyelocytic leukemia cells, Daxx does not localize to PODs. However, upon treatment with retinoic acid, which induces disease remission, Daxx relocalizes to PODs (33). Despite evidence supporting a proapoptotic function for Daxx, other studies have demonstrated that Daxx is essential to cell survival or is antiapoptotic. Targeted disruption of Daxx in mice results in embryonic lethality accompanied by extensive apoptosis (21). Elevated apoptosis was also observed in Daxx-null embryonic stem cells (21) as well as in fibroblasts in which endogenous Daxx was depleted by RNA interference treatment (J. S. Michaelson and P. Leder, unpublished data). In myeloid precursor cells, Daxx overexpression inhibited activated cell death, indicating an antiapoptotic role for Daxx (2). Furthermore, an antiapoptotic role for Daxx in acute promyelocytic leukemia cells was proposed, as Daxx expression decreased after apoptotic induction with HDAC1 inhibitors (1). It is possible that Daxx provides bipartite functions. Under certain circumstances, Daxx may be essential for cell survival, and under other circumstances, Daxx may propagate apoptotic signals. Similar to the HIPKs, Daxx functions as a transcriptional regulator. For example, Daxx repressed the transcriptional activities of the Pax3 and ETS1 transcription factors (11, 18). Interestingly, Daxx was not able to repress the transcriptional activities of the oncogenic fusion protein Pax3-FKHR present in an alveolar rhabdomyosarcoma. This suggests that Pax3-FKHR circumvents the transcriptional controls normally applied to Pax3 (11). Daxx also either repressed or activated Pax5-mediated transcription (6). The specific effect of Daxx on Pax5 activity varied in different B-cell lines. Activation of Pax5-mediated transcription by Daxx depended on recruitment of the histone acetyltransferase CREB binding protein. To date, the regulation of Daxx transcriptional activity is poorly understood. It was suggested that in the absence of PML, Daxx localized to chromatin, where it recruited HDAC1 and repressed transcription (12, 17). Overexpression of PML but not the oncogenic fusion PML-retinoic acid receptor α recruits Daxx to PODs, thereby inhibiting Daxx repression (16, 17). The recruitment of Daxx from chromatin to PODs requires the secondary modification of PML by the ubiquitin-like molecule SUMO-1 (12, 16, 17). These results demonstrate that Daxx transcriptional regulatory activity is controlled in part by PML, which sequesters Daxx from condensed chromatin to PODs. In this study, we characterized the expression and localization of HIPK1. In addition, we propose two novel mechanisms for regulating Daxx behavior, both mediated by HIPK1. HIPK1 relocates Daxx from PODs, presumably to chromatin, where Daxx participates in transcriptional regulation. In addition, HIPK1 phosphorylates Daxx on Ser 669, a site important in modulating Daxx activity.