The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization

Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.     

Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2

The serine/threonine kinase HIPK2 phosphorylates the p53 protein at Ser 46, thus promoting p53-dependent gene expression and subsequent apoptosis. Here, we show that DNA damaging chemotherapeutic drugs cause degradation of endogenous HIPK2 dependent on the presence of a functional p53 protein. Early induced p53 allows caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977. The resulting C-terminally truncated HIPK2 forms show an enhanced induction of the p53 response and cell death, thus allowing the rapid amplification of the p53-dependent apoptotic program during the initiation phase of apoptosis by a regulatory feed-forward loop. The active HIPK2 fragments are further degraded during the execution and termination phase of apoptosis, thus ensuring the occurrence of HIPK2 signaling only during the early phases of apoptosis induction.     

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.     

Covalent modification of human homeodomain interacting protein kinase 2 by SUMO-1 at lysine 25 affects its stability

The HIPK2 protein is a critical regulator of apoptosis and functionally interacts with p53 to increase gene expression. Here we show that human HIPK2 is modified by sumoylation at lysine 25, as revealed by in vivo and in vitro experiments. While SUMO-1 modification of HIPK2 has no influence on its ability to phosphorylate p53 at serine 46, to induce gene expression, and to mediate apoptosis, a non-sumoylatable HIPK2 mutant displays a strongly increased protein stability. The N-terminal SUMO-1 modification site is conserved between all vertebrate HIPK2 proteins and is found in all members of the HIPK family of protein kinases. Accordingly, also human HIPK3 is modified by sumoylation.     

DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.     

HIPK2: A tumour suppressor that controls DNA damage‐induced cell fate and cytokinesis

In response to DNA‐damage, cells have to decide between different cell fate programmes. Activation of the tumour suppressor HIPK2 specifies the DNA damage response (DDR) and tips the cell fate balance towards an apoptotic response. HIPK2 is activated by the checkpoint kinase ATM, and triggers apoptosis through regulatory phosphorylation of a set of cellular key molecules including the tumour suppressor p53 and the anti‐apoptotic corepressor CtBP. Recent work has identified HIPK2 as a regulator of the ultimate step in cytokinesis: the abscission of the mother and daughter cells. Since proper cytokinesis is essential for genome stability and maintenance of correct ploidy, this finding sheds new light on the tumour suppressor function of HIPK2. Here we highlight the molecular mechanisms coordinating HIPK2 function and discuss its emerging role as a tumour suppressor.     

Avian Reovirus activates a novel proapoptotic signal by linking Src to p53

We have previously shown that avian reovirus (ARV) S1133 and its structural protein σC cause apoptosis in cultured Vero cells through an unknown intracellular signaling pathway. This work investigates how ARV S1133 induces proapoptotic signals. Upon ARV S1133 infection and subsequent apoptosis, levels of p53 mRNA and protein, and p53 serine-46 and serine-392 phosphorylation increased. In addition, p53-driven reporter activity and levels of the p53-induced apoptotic protein bax were increased, and Src tyrosine-418 phosphorylation was elevated. UV-inactivated virus failed to activate Src, p53 or induce apoptosis. Over-expression of dominant negative p53, or treatment with tyrosine kinase inhibitor genistein protected cells from ARV S1133-induced apoptosis. Inhibition of Src by over-expression of C-terminal Src kinase (Csk) or treatment with Src family tyrosine kinase inhibitor SU-6656 diminished the ARV S1133-induced p53 expression, activation, and apoptosis. Over-expression of σC resulted in the upregulation of p53, p53 serine-46 phosphorylation, p53-driven reporter activity and accumulation of bax. σC expression during ARV S1133 infection was concomitant with the onset of apoptosis. These studies provide strong evidence that the viral gene expression is required for ARV S1133 to initiate a proapoptotic signal via Src to p53. In addition, σC was able to utilize a p53-dependent pathway to elicit apoptosis.