Homeodomain-interacting Protein Kinase 2 Degradation Research Articles

Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy.

Phosphorylated methyl-CpG binding protein2 (p-MeCP2) suppresses the processing of several microRNAs (miRNAs). Homeo-domain interacting protein kinase2 (HIPK2) phosphorylates MeCP2, a known transcriptional repressor. However, it is not known if MeCP2 and HIPK2 are involved in processing of miRNAs implicated in diabetic nephropathy. p-MeCP2 and HIPK2 levels were significantly increased, but Seven in Absentia Homolog1 (SIAH1), which mediates proteasomal degradation of HIPK2, was decreased in the glomeruli of streptozotocin injected diabetic mice. Among several miRNAs, miR-25 and its precursor were significantly decreased in diabetic mice, whereas primary miR-25 levels were significantly increased. NADPH oxidase4 (NOX4), a target of miR-25, was significantly increased in diabetic mice. Protein levels of p-MeCP2, HIPK2, and NOX4 were increased in high glucose (HG)- or TGF-β-treated mouse glomerular mesangial cells (MMCs). miR-25 (primary, precursor, and mature) and mRNA levels of genes indicated in the in vivo study showed similar trends of regulation in MMCs treated with HG or TGF-β. The HG- or TGF-β-induced upregulation of p-MeCP2, NOX4 and primary miR-25, but downregulation of precursor and mature miR-25, were attenuated by Hipk2 siRNA. These results demonstrate a novel role for the SIAH1/HIPK2/MeCP2 axis in suppressing miR-25 processing and thereby upregulating NOX4 in early diabetic nephropathy.

Hyperglycemia triggers HIPK2 protein degradation.

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.

PARP1 regulates the protein stability and proapoptotic function of HIPK2.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2.

Interplay between Mdm2 and HIPK2 in the DNA damage response.

The tumour suppressor p53 is activated to induce cell-cycle arrest or apoptosis in the DNA damage response (DDR). p53 phosphorylation at Ser46 by HIPK2 (homeodomain-interacting protein kinase 2) is a critical event in apoptosis induction. Interestingly, HIPK2 is degraded by Mdm2 (a negative regulator of p53), whereas Mdm2 is downregulated by HIPK2 through several mechanisms. Here, we develop a four-module network model for the p53 pathway to clarify the role of interplay between Mdm2 and HIPK2 in the DDR evoked by ultraviolet radiation. By numerical simulations, we reveal that Mdm2-dependent HIPK2 degradation promotes cell survival after mild DNA damage and that inhibition of HIPK2 degradation is sufficient to trigger apoptosis. In response to severe damage, p53 phosphorylation at Ser46 is promoted by the accumulation of HIPK2 due to downregulation of nuclear Mdm2 in the later phase of the response. Meanwhile, the concentration of p53 switches from moderate to high levels, contributing to apoptosis induction. We show that the presence of three mechanisms for Mdm2 downregulation, i.e. repression of mdm2 expression, inhibition of its nuclear entry and HIPK2-induced degradation, guarantees the apoptosis of irreparably damaged cells. Our results agree well with multiple experimental observations, and testable predictions are also made. This work advances our understanding of the regulation of p53 activity in the DDR and suggests that HIPK2 should be a significant target for cancer therapy.

SIRT1 negatively regulates the protein stability of HIPK2

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.

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.

A systems approach identifies HIPK2 as a key regulator of kidney fibrosis

Kidney fibrosis is a common process that leads to the progression of kidney diseases. We used an integrated computational/experimental systems biology approach to identify upstream protein kinases that regulate gene expression changes in kidneys of HIV-1 transgenic mice (Tg26), which have both tubulo-interstitial fibrosis and glomerulosclerosis. We identified the homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in kidneys of Tg26 and patients with various kidney diseases. HIV infection increased the protein level of HIPK2 by promoting oxidative stress, which inhibited SIAH1-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and expression of epithelial-mesenchymal trans-differentiation markers in kidney epithelial cells by activating p53, TGF-β/Smad3, and Wnt/Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 as well as in other animal models of kidney fibrosis. We conclude that HIPK2 is a potential target for anti-fibrosis therapy.

HIF-1α antagonizes p53-mediated apoptosis by triggering HIPK2 degradation

Many human diseases are characterized by the development of tissue hypoxia. Hypoxia-inducible factor (HIF) is a transcription factor that regulates fundamental cellular processes in response to changes in oxygen concentration, such as angiogenesis, survival, and alterations in metabolism. The levels of HIF-1α subunit are increased in most solid tumors not only by low oxygen but also by growth factors and oncogenes and correlate with patient prognosis and treatment failure. The link between HIF-1α and apoptosis, a major determinant of cancer progression and treatment outcome, is poorly understood. Here we show that HIF-1α protects against drug-induced apoptosis by antagonizing the function of the tumor suppressor p53. HIF-1α upregulation induced proteasomal degradation of homeodomain-interacting protein kinase-2 (HIPK2), the p53 apoptotic activator. Inhibition of HIF-1α by siRNA, HIF-1α-dominant negative or by zinc re-established the HIPK2 levels and the p53-mediated chemosensitivity in tumor cells. Our findings identify a novel circuitry between HIF-1α and p53, and provide a paradigm for HIPK2 dictating cell response to antitumor therapies.

Unfolded p53 in the pathogenesis of Alzheimer's disease: is HIPK2 the link?

p53 transcriptional activity depends mainly on posttranslational modifications and protein/protein interaction. Another important mechanism that controls p53 function is its conformational stability since p53 is an intrinsically unstable protein. An altered conformational state of p53, independent from point mutations, has been reported in tissues from patients with Alzheimer's disease (AD), leading to an impaired and dysfunctional response to stressors. Recent evidence shows that one of the activators that induces p53 posttranslational modification and wild-type conformational stability is homeodomain interacting protein kinase 2 (HIPK2). Hence, conditions that induce HIPK2 deregulation would result in a dysfunctional response to stressors by affecting p53 activity. Discovering the mechanisms of HIPK2 activation/inhibition and the ways to manipulate HIPK2 activity are an interesting option to affect several biological pathways, including those underlying AD. Soluble beta-amyloid peptides have recently been involved in HIPK2 degradation, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus, before triggering the amyloidogenic cascade. Here we discuss about these findings and the potential relevance of HIPK2 as a target for AD and highlight the existence of a novel amyloid-based mechanism in AD potentially leading to the survival of injured dysfunctional cells.

Counteracting MDM2-induced HIPK2 downregulation restores HIPK2/p53 apoptotic signaling in cancer cells

Homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating serine 46 (Ser46) in response to DNA damage. In tumors with wild-type p53, its tumor suppressor function is often impaired by MDM2 overexpression that targets p53 for proteasomal degradation. Likewise, MDM2 targets HIPK2 for protein degradation impairing p53-apoptotic function. Here we report that zinc antagonised MDM2-induced HIPK2 degradation as well as p53 ubiquitination. The zinc inhibitory effect on MDM2 activity leads to HIPK2-induced p53Ser46 phosphorylation and p53 pro-apoptotic transcriptional activity. These results suggest that zinc derivatives are potential molecules to target the MDM2-induced HIPK2/p53 inhibition.

Homeodomain Interacting Protein Kinase 2: A Target for Alzheimer's Beta Amyloid Leading to Misfolded p53 and Inappropriate Cell Survival

BackgroundHomeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase whose activity is fundamental in maintaining wild-type p53 function, thereby controlling the destiny of cells when exposed to DNA damaging agents. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer's Disease (AD) that led to an impaired and dysfunctional response to stressors.Methodology/Principal FindingsHere we examined the molecular mechanisms underlying the impairment of p53 activity in two cellular models, HEK-293 cells overexpressing the amyloid precursor protein and fibroblasts from AD patients, starting from recent findings showing that p53 conformation may be regulated by HIPK2. We demonstrated that beta-amyloid 1–40 induces HIPK2 degradation and alters HIPK2 binding activity to DNA, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus. Expression of HIPK2 was analysed by western blot experiments, whereas HIPK2 DNA binding was examined by chromatin immunoprecipitation analysis. In particular, we evaluated the recruitment of HIPK2 onto some target promoters, including hypoxia inducible factor-1α and metallothionein 2A.Conclusions/SignificanceThese results support the existence of a novel amyloid-based pathogenetic mechanism in AD potentially leading to the survival of injured dysfunctional cells.

Hypoxia suppresses chemotherapeutic drug-induced p53 Serine 46 phosphorylation by triggering HIPK2 degradation

The molecular mechanisms by which hypoxic tumor cells escape radio- and chemotherapy are largely unclear. Homeodomain-interacting protein kinase 2 (HIPK2) drives the apoptotic program in response to DNA-damaging chemotherapeutic drug treatment by phosphorylating the tumor suppressor protein p53 at Ser46. HIPK2 is kept inactive in unstressed cells through ubiquitination and degradation facilitated by the ubiquitin ligases WSB1 and Siah1. Here, we demonstrate that HIPK2 is degraded during hypoxia in a proteasome-dependent and partially Siah1-dependent fashion. Concordantly, hypoxic tumor cells show an impaired p53 Ser46 phosphorylation in response to treatment with the chemotherapeutic Adriamycin. Remarkably, proteasome-inhibition rescues HIPK2 expression in hypoxic hepatoma cells and restores p53 Ser46 phosphorylation and caspase activity after Adriamycin treatment. Our findings suggest a molecular mechanism by which hypoxic cancer cells can escape chemotherapeutic drug treatment and suggest proteasome-inhibition as a promising approach to sensitise hypoxic cancer cells to therapy.

Stabilization of HIPK2 by escape from proteasomal degradation mediated by the E3 ubiquitin ligase Siah1

Homeodomain-interacting protein kinase 2 (HIPK2) induces apoptosis and, thus, is maintained at a low level via ubiquitin-mediated proteolysis. In a yeast two-hybrid screen, we identified Siah1, a RING finger E3 ubiquitin ligase, as an interacting protein of HIPK2. Siah1 targeted HIPK2 for poly-ubiquitination-mediated proteasomal degradation. Degradation of HIPK2 by Siah1 was blocked by forced expression of either Mixed Lineage Kinase-3 or Epstein-Barr viral protein LMP-1, as well as by DNA damaging stimuli. These findings effectively illustrate the regulatory mechanisms underlying HIPK2 stabilization by escape from Siah1-mediated degradation, and that Siah1 is an integration target for several internal or external stimuli for HIPK2 stabilization.

How cells switch HIPK2 on and off

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging regulator of cell growth and apoptosis in various cell types, tissues and organisms. Previous work indicates that HIPK2 is a potential tumour suppressor and DNA damage-responsive kinase, which phosphorylation-dependently activates the apoptotic programme by engaging diverse downstream targets, including tumour suppressor p53 and the anti-apoptotic transcriptional corepressor C-terminal binding protein. The regulation of HIPK2, however, remained largely obscure. Recent studies show that HIPK2 activity is mainly controlled at the post-transcriptional level through targeted proteolysis. Caspase-dependent processing triggers HIPK2 hyperactivation, whereas the ubiquitin-proteasome system (UPS) keeps HIPK2 in check by targeting it for degradation. Both HIPK2 hyperactivation and HIPK2 degradation are under the control of transcription factor p53. Negative regulation of HIPK2 by the UPS is abolished in response to DNA damage, which facilitates HIPK2 stabilization and activation. Here we discuss these findings in the context of DNA damage signalling and tumour suppression.

Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.

Ubiquitination and Degradation of Homeodomain‐interacting Protein Kinase 2 (HIPK2) by WSB‐1

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of nuclear protein kinases family that act as cofactors for various transcription factors. HIPK2 was induced by UV irradiation and cisplatin treatment, implying degradation of HIPK2 in normal cells. Here we show that HIPK2 is ubiquitinated and degraded by WD40-repeat/SOCS box protein WSB-1, which process is blocked by UV irradiation. Yeast two-hybrid screen was performed to identify interacting proteins of HIPK2. Ube2d-1 and WSB-1 were characterized as HIPK2 interacting protein, which is E2 ubiquitin-conjugating enzyme and ubiquitin E3 ligase, respectively. Coexpression of WSB-1 resulted in degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 revealed that WD40 repeats were sufficient for its interaction with and degradation of HIPK2, whereas SOCS box did not involved in degradation of HIPK2. In support of HIPK2 degradation by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. Ubiquitination of HIPK2 was observed by incubation of HIPK2 with immunoprecipitated WSB-1 complexes in vitro, but not with purified WSB-1, suggesting that WSB-1 forms a complex with other proteins to act as an E3 ubiquitin ligase. Ubiquitination and degradation of HIPK2 by WSB-1 was completely inhibited by UV irradiation and cisplatin treatment. These findings strongly illustrate regulatory mechanisms by which HIPK2 is maintained low level by WSB-1 in normal cells, and stabilized by genotoxic stresses, such as UV and cisplatin.