ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury.

In this study we investigate the role of Zipper-interacting protein kinase (ZIPK) in high glucose-induced vascular injury, focusing on its interaction with STAT5A and its effects on p53 and inducible nitric oxide synthase (NOS2) expression. Human umbilical vein endothelial cells (HUVECs) are cultured under normal (5 mM) and high (25 mM) glucose conditions. Protein and gene expression levels are assessed by western blot analysis and qPCR respectively, while ROS levels are measured via flow cytometry. ZIPK expression is manipulated using overexpression plasmids, siRNAs, and shRNAs. The effects of the ZIPK inhibitor TC-DAPK6 are evaluated in a diabetic rat model. Our results show that high glucose significantly upregulates ZIPK, STAT5A, p53, and NOS2 expressions in HUVECs, thus increasing oxidative stress. Silencing of STAT5A reduces p53 and NOS2 expressions and reactive oxygen species (ROS) accumulation. ZIPK is essential for high glucose-induced p53 expression and ROS accumulation, while silencing of ZIPK reverses these effects. Overexpression of ZIPK combined with STAT5A silencing attenuates glucose-induced alterations in p53 and NOS2 expression, thereby preventing cell damage. Coimmunoprecipitation reveals a direct interaction between ZIPK and STAT5A in the nucleus under high-glucose condition. In diabetic rats, TC-DAPK6 treatment significantly decreases ZIPK, p53, and NOS2 expressions. Our findings suggest that ZIPK plays a critical role in high glucose-induced vascular injury via STAT5A-mediated pathways, proposing that ZIPK is a potential therapeutic target for diabetic vascular complications.

Zipper interacting protein kinase (ZIPK) is a negative regulator of HIV-1 replication that is restricted by viral Nef protein through proteasomal degradation.

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.

ZIP kinase phosphorylated and activated by Rho kinase/ROCK contributes to cytokinesis in mammalian cultured cells.

Cytokinesis of animal cells requires contraction of a contractile ring, composed of actin filaments and myosin II filaments. Phosphorylation of myosin II regulatory light chain (MRLC) promotes contraction of the actomyosin ring by activating myosin II motor activity. Both Rho-associated coiled-coil kinase (Rho kinase/ROCK) and Zipper-interacting protein kinase (ZIP kinase/ZIPK) have been reported to phosphorylate MRLC at the contractile ring. However, it remains unclear whether these kinases function independently of each other. Here, we clarified that ROCK colocalizes and forms a complex with ZIPK at telophase. As ROCK is reported to phosphorylate and activate ZIPK in vitro, we hypothesized that ZIPK phosphorylated by ROCK contributes to control cytokinesis. To address this, we expressed EGFP-ZIPK wild type (WT), a non-phosphorylatable mutant (T265A) or a phosphorylation-mimicking mutant (T265D) in HeLa cells and treated these cells with a ROCK inhibitor. Decrease in phosphorylated MRLC and a delay of furrow ingression by the ROCK inhibitor were rescued by the expression of EGFP-ZIPK-T265D, but not EGFP-ZIPK-WT or -T265A. This suggests that ROCK regulates MRLC phosphorylation followed by furrow ingression, through ZIPK phosphorylation.

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells.

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells.

Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.

Validation of chemical genetics for the study of zipper-interacting protein kinase signaling.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr kinase that mediates a variety of cellular functions. Analogue-sensitive kinase technology was applied to the study of ZIPK signaling in coronary artery smooth muscle cells. ZIPK was engineered in the ATP-binding pocket by substitution of a bulky gatekeeper amino acid (Leu93) with glycine. Cell-permeable derivatives of pyrazolo[3,4-d]pyrimidine provided effective inhibition of L93G-ZIPK (1NM-PP1, IC50 , 1.0 µM; 3MB-PP1, IC50 , 2.0 µM; and 1NA-PP1, IC50 , 8.6 µM) but only 3MB-PP1 had inhibitory potential (IC50 > 10 µM) toward wild-type ZIPK. Each of the compounds also attenuated Rho-associated coiled-coil containing protein kinase (ROCK) activity under experimental conditions found to be optimal for inhibition of L93G-ZIPK. In silico molecular simulations showed effective docking of 1NM-PP1 into ZIPK following mutational enlargement of the ATP-binding pocket. Molecular simulation of 1NM-PP1 docking in the ATP-binding pocket of ROCK was also completed. The 1NM-PP1 inhibitor was selected as the optimal compound for selective chemical genetics in smooth muscle cells since it displayed the highest potency for L93G-ZIPK relative to WT-ZIPK and the weakest off-target effects against other relevant kinases. Finally, the 1NM-PP1 and L93G-ZIPK pairing was effectively applied in vascular smooth muscle cells to manipulate the phosphorylation level of LC20, a previously defined target of ZIPK.

The effect of aspartame on accelerating caspase-dependent apoptosis of pancreatic islet via ZIPK/STAT3/caspase 3 signaling pathway.

Aspartame (ASP) as an important sugar substitute is widely used in pharmaceutical and food processing. Here, we compared the effects of ASP and sucrose on mice pancreatic islet cells in vivo and observed that ASP with the condition of high concentration and long-term exposure (HASP) could cause insulin secretion (500 mg/kg for 1 month). Next, we conducted iTRAQ mass spectrometry to profile the global phosphoproteome and found that phosphorylation of zipper-interacting protein kinase (ZIPK) in murine pancreatic islet tissues were induced at Thr197, Thr242, Thr282, and Ser328 by high-sucrose (HS) treatment, but only induced at Thr197 and Ser328 by HASP treatment. Simultaneously, phosphorylation of STAT3 could be induced at Tyr705 and Ser727 by HS but not by HASP. Furthermore, presence of activated STAT3 accompanied with autophagy was observed in HS treatment. In turn, the inactivation of STAT3 as well as enhanced expression of caspase 3 was observed in HASP treatment. We generated Thr242APro and Thr282Pro on ZIPK using CRISPR-Cas9 in ß-TC3 cells and found the weakened interaction with STAT3 as well as the reduced phosphorylation of STAT3 even under HS stimulation. Finally, we observed that ankyrin repeat domain containing 11 (ANKRD11) could interact with ZIPK and play an inhibitory role in the phosphorylation of Thr242APro and Thr282Pro of ZIPK. However, HASP can induce the retention of ANKRD11 in the cytoplasm by phenylpyruvic acid (the metabolite of ASP). Taken together, this study determined that ASP with high concentration and long-term exposure could lead to caspase-dependent apoptosis of pancreatic islet cells through ANKRD11/ZIPK/STAT3 inhibition. Our results give evidence of adverse effects of aspartame on islet cells in some extreme conditions, which might help people to reconsider the biosafety of non-nutritive sweeteners.

Organ-specific off-target effects of Pim/ZIP kinase inhibitors suggest lack of contractile Pim kinase activity in prostate, bladder, and vascular smooth muscle.

Smooth muscle contraction by Pim kinases and ZIPK has been suggested, but evidence for lower urinary tract organs or using Pim-selective inhibitor concentrations is not yet available. Here, we assessed effects of the Pim inhibitors AZD1208 and TCS PIM-1 and the dual ZIPK/Pim inhibitor HS38 on contractions of human prostate and bladder tissues and of porcine interlobar arteries. Human tissues were obtained from radical prostatectomy and radical cystectomy and renal interlobar arteries from pigs. Contractions were studied in an organ bath. Noradrenaline-, phenylephrine- and methoxamine-induced contractions were reduced (up to > 50%) with 500-nM AZD1208 in prostate tissues and to lesser degree and not consistently with all agonists in interlobar arteries. A total of 100-nM AZD1208 or 500-nM TCS PIM-1 did not affect agonist-induced contractions in prostate tissues. Decreases in agonist-induced contractions with 3-µM HS38 in prostate tissues and interlobar arteries were of small extent and did not occur with each agonist. Carbachol-induced contractions in detrusor tissues were unchanged with AZD1208 (500 nM) or HS38. Electric field stimulation-induced contractions were not affected with AZD1208 or HS38 in any tissue, but slightly reduced with 500-nM TCS PIM-1 in prostate tissues. Concentration-dependent effects of Pim inhibitors suggest lacking Pim-driven smooth muscle contraction in the prostate, bladder, and interlobar arteries but point to organ-specific functions of off-targets. Procontractile functions of ZIPK in the prostate and interlobar arteries may be limited and are lacking in the detrusor.

Substrate stiffness induces nuclear localization of myosin regulatory light chain to suppress apoptosis.

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.

VIRMA contributes to non-small cell lung cancer progression via N6-methyladenosine-dependent DAPK3 post-transcriptional modification.

N6-methyladenosine (m6A) has been reported to be abnormally expressed in non-small cell lung cancer (NSCLC), and plays a vital role in regulation of cell proliferation, invasion and metastasis. Vir-Like m6A methyltransferase associated (VIRMA, also called KIAA1429) has not been well studied in NSCLC. Thus, in this study, we investigated the biological impact and underlying mechanism of VIRMA in NSCLC. High expression of VIRMA was testified in patients with NSCLC and predicted worse prognosis in patients. VIRMA facilitated cell proliferation and tumor growth both in vitro and in vivo. Furthermore, VIRMA-regulated m6A modifications led to post-transcriptional suppression of death-associated protein kinase 3 (DAPK3, also called ZIP or ZIPK) through the YT521-B homology domain-containing family proteins 2/3(YTHDF2/3). Inhibition of DAPK3 rescued the tumor-suppressive phenotypes induced by VIRMA deficiency. In conclusion, VIRMA-guided m6A modifications promoted NSCLC progression via m6A-dependent degradation of DAPK3 mRNA. Therefore, VIRMA may be a novel therapeutic target in NSCLC.

Analyzing Recombinant Protein Production in Pichia pastoris with Targeted Proteomics.

New mass spectrometry approaches enable antibody-independent tracking of protein production. Herein, we outline an antibody-independent mass spectrometry method for tracking recombinant protein production in the methylotrophic yeast Pichia pastoris system.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Systems biology network-based discovery of a small molecule activator BL-AD008 targeting AMPK/ZIPK and inducing apoptosis in cervical cancer.

The aim of this study was to discover a small molecule activator BL-AD008 targeting AMPK/ZIPK and inducing apoptosis in cervical cancer. In this study, we systematically constructed the global protein-protein interaction (PPI) network and predicted apoptosis-related protein connections by the Naïve Bayesian model. Then, we identified some classical apoptotic PPIs and other previously unrecognized PPIs between apoptotic kinases, such as AMPK and ZIPK. Subsequently, we screened a series of candidate compounds targeting AMPK/ZIPK, synthesized some compounds and eventually discovered a novel dual-target activator (BL-AD008). Moreover, we found BL-AD008 bear remarkable anti-proliferative activities toward cervical cancer cells and could induce apoptosis by death-receptor and mitochondrial pathways. Additionally, we found that BL-AD008-induced apoptosis was affected by the combination of AMPK and ZIPK. Then, we found that BL-AD008 bear its anti-tumor activities and induced apoptosis by targeting AMPK/ZIPK in vivo. In conclusion, these results demonstrate the ability of systems biology network to identify some key apoptotic kinase targets AMPK and ZIPK; thus providing a dual-target small molecule activator (BL-AD008) as a potential new apoptosis-modulating drug in future cervical cancer therapy.

The effect of aspartame on accelerating caspase-dependent apoptosis of pancreatic islet via ZIPK/STAT3/caspase 3 signaling pathway

Aspartame (ASP) as an important sugar substitute is widely used in pharmaceutical and food processing. Here, we compared the effects of ASP and sucrose on mice pancreatic islet cells in vivo and observed that ASP with the condition of high concentration and long-term exposure (HASP) could cause insulin secretion (500 mg/kg for 1 month). Next, we conducted iTRAQ mass spectrometry to profile the global phosphoproteome and found that phosphorylation of zipper-interacting protein kinase (ZIPK) in murine pancreatic islet tissues were induced at Thr197, Thr242, Thr282, and Ser328 by high-sucrose (HS) treatment, but only induced at Thr197 and Ser328 by HASP treatment. Simultaneously, phosphorylation of STAT3 could be induced at Tyr705 and Ser727 by HS but not by HASP. Furthermore, presence of activated STAT3 accompanied with autophagy was observed in HS treatment. In turn, the inactivation of STAT3 as well as enhanced expression of caspase 3 was observed in HASP treatment. We generated Thr242APro and Thr282Pro on ZIPK using CRISPR-Cas9 in β-TC3 cells and found the weakened interaction with STAT3 as well as the reduced phosphorylation of STAT3 even under HS stimulation. Finally, we observed that ankyrin repeat domain containing 11 (ANKRD11) could interact with ZIPK and play an inhibitory role in the phosphorylation of Thr242APro and Thr282Pro of ZIPK. However, HASP can induce the retention of ANKRD11 in the cytoplasm by phenylpyruvic acid (the metabolite of ASP). Taken together, this study determined that ASP with high concentration and long-term exposure could lead to caspase-dependent apoptosis of pancreatic islet cells through ANKRD11/ZIPK/STAT3 inhibition. Our results give evidence of adverse effects of aspartame on islet cells in some extreme conditions, which might help people to reconsider the biosafety of non-nutritive sweeteners. (AU)

The effect of aspartame on accelerating caspase-dependent apoptosis of pancreatic islet via ZIPK/STAT3/caspase 3 signaling pathway

Aspartame (ASP) as an important sugar substitute is widely used in pharmaceutical and food processing. Here, we compared the effects of ASP and sucrose on mice pancreatic islet cells in vivo and observed that ASP with the condition of high concentration and long-term exposure (HASP) could cause insulin secretion (500 mg/kg for 1 month). Next, we conducted iTRAQ mass spectrometry to profile the global phosphoproteome and found that phosphorylation of zipper-interacting protein kinase (ZIPK) in murine pancreatic islet tissues were induced at Thr197, Thr242, Thr282, and Ser328 by high-sucrose (HS) treatment, but only induced at Thr197 and Ser328 by HASP treatment. Simultaneously, phosphorylation of STAT3 could be induced at Tyr705 and Ser727 by HS but not by HASP. Furthermore, presence of activated STAT3 accompanied with autophagy was observed in HS treatment. In turn, the inactivation of STAT3 as well as enhanced expression of caspase 3 was observed in HASP treatment. We generated Thr242APro and Thr282Pro on ZIPK using CRISPR-Cas9 in β-TC3 cells and found the weakened interaction with STAT3 as well as the reduced phosphorylation of STAT3 even under HS stimulation. Finally, we observed that ankyrin repeat domain containing 11 (ANKRD11) could interact with ZIPK and play an inhibitory role in the phosphorylation of Thr242APro and Thr282Pro of ZIPK. However, HASP can induce the retention of ANKRD11 in the cytoplasm by phenylpyruvic acid (the metabolite of ASP). Taken together, this study determined that ASP with high concentration and long-term exposure could lead to caspase-dependent apoptosis of pancreatic islet cells through ANKRD11/ZIPK/STAT3 inhibition. Our results give evidence of adverse effects of aspartame on islet cells in some extreme conditions, which might help people to reconsider the biosafety of non-nutritive sweeteners. (AU)