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.     

Cutaneous HPV23 E6 prevents p53 phosphorylation through interaction with HIPK2

Ultraviolet irradiation (UV) is the major risk factor for the development of skin cancer. Moreover, increasing evidence suggests cutaneotropic human papillomaviruses (HPV) from the beta genus to play a causal role as a co-factor in the development of cutaneous squamous cell carcinoma. Homeodomain-interacting protein kinase 2 (HIPK2) operates as a potential suppressor in skin tumorigenesis and is stabilized by UV-damage. HIPK2 is an important regulator of apoptosis, which forms a complex with the tumor suppressor p53, mediating p53 phosphorylation at Ser 46 and thus promoting pro-apoptotic gene expression. In our study, we demonstrate that cutaneous HPV23 E6 protein directly targets HIPK2 function. Accordingly, HPV23 E6 interacts with HIPK2 both in vitro and in vivo. Furthermore, upon massive UVB-damage HPV23 E6 co-localizes with endogenous HIPK2 at nuclear bodies. Functionally, we demonstrate that HPV23 E6 inhibits HIPK2-mediated p53 Ser 46 phosphorylation through enforcing dissociation of the HIPK2/p53 complex. In addition, HPV23 E6 co-accumulates with endogenous HIPK2 upon UV damage suggesting a mechanism by which HPV23 E6 keeps HIPK2 in check after UV damage. Thus, cutaneous HPV23 E6 prevents HIPK2-mediated p53 Ser 46 phosphorylation, which may favour survival of UV-damaged keratinocytes and skin carcinogenesis by apoptosis evasion.     

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.     

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.     

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.     

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.     

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.     

MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.     

MDM2 Inhibits Axin-Induced p53 Activation Independently of its E3 Ligase Activity

MDM2 plays a crucial role in negatively regulating the functions of tumor suppressor p53. Here we show that MDM2 can inhibit Axin-stimulated p53-dependent apoptosis by suppressing p53 phosphorylation at Ser 46 and apoptosis-related p53 transactivational activity. Interestingly, the ubiquitin E3 ligase activity of MDM2 is not required for this inhibitory effect. Mechanically, either wildtype MDM2 or its E3-dead mutant, disrupts the Axin-based HIPK2/p53 complex formation by blocking the binding of p53 and HIPK2 to Axin. MDM2Δp53, a deletion mutant that lacks p53 binding domain fails to exert the inhibitory effect, demonstrating that the interaction of MDM2 and p53, but not its E3 ligase activity toward p53 plays key role in suppressing Axin-stimulated p53 activation. Our results thus have revealed a novel aspect of the mechanism by which MDM2 regulates p53 activities.