ZIP Kinase Research Articles

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-β induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-β significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-β stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-β significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-β. ZIPK gene knockdown (KD) also significantly reduced TGF-β-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-β-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

Clostridiumnovyi's Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells.

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

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.

Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells

Zipper-interacting protein kinase (ZIPK) is known to regulate several functions such as apoptosis, smooth muscle contraction, and cell migration. While exogenously expressed GFP-ZIPK localizes to the cleavage furrow, role of ZIPK in cytokinesis is obscure. Here, we show that ZIPK is a major MRLC kinase during mitosis. Moreover, ZIPK siRNA-mediated knockdown causes delay of cytokinesis. The delay in cytokinesis of ZIPK-knockdown cells was rescued by the exogenous diphosphorylation-mimicking MRLC mutant. Taken together, these findings suggest that ZIPK plays a role in the progression and completion of cytokinesis through MRLC phosphorylation.

Unc-51 like kinase 1 (ULK1) in silico analysis for biomarker identification: A vital component of autophagy

Autophagy is a degradation pathway involving lysosomal machinery for degradation of damaged organelles like the endoplasmic reticulum and mitochondria into their building blocks to maintain homeostasis within the cell. ULK1, a serine/threonine kinase, is conserved across species, from yeasts to mammals, and plays a central role in autophagy pathway. It receives signals from upstream modulators such as TIP60, mTOR and AMPK and relays them to its downstream substrates like Ambra1 and ZIP kinase. The activity of this complex is regulated through protein–protein interactions and post-translational modifications. Applying in silico analysis we identified (i) conserved patterns of ULK1 that showed its evolutionary relationship between the species which were closely related in a family compared to others. (ii) A total of 23 TFBS distributed throughout ULK1 and nuclear factor (erythroid-derived) 2 (NFE2) is of utmost significance because of its high importance rate. NEF2 has already been shown experimentally to play a role in the autophagy pathway. Most of these were of zinc coordinating class and we suggest that this information could be utilized to modulate this pathway by modifying interactions of these TFs with ULK1. (iii) CATTT haplotype was prominently found with frequency 0.774 in the studied population and nsSNPs which could have harmful effect on ULK1 protein and these could further be tested. (iv) A total of 83 phosphorylation sites were identified; 26 are already known and 57 are new that include one at tyrosine residue which could further be studied for its involvement in ULK1 regulation and hence autophagy. Furthermore, 4 palmitoylation sites at positions 426, 927, 1003 and 1049 were also found which could further be studied for protein–protein interactions as well as in trafficking.

Spinal protein kinase Mζ contributes to the maintenance of peripheral inflammation-primed persistent nociceptive sensitization after plantar incision.

Previous studies suggest that persistent post-surgical pain (PPSP) is correlated with preoperative pain status and amplification of central sensitization. Protein kinase Mζ (PKMζ) is an essential substrate of the late long-term potentiation underlying central sensitization, which is one mechanism of pain memory formation. However, the potential contributions of spinal PKMζ to PPSP, a condition in which preoperative pain is prevalent, are not known. Here, a modified 'hyperalgesia priming' model was established to simulate the clinical situation. This model used intraplantar injections of carrageenan (Car) as priming stimuli to elicit persistent nociceptive sensitization after plantar incision in rats. Upon treatment with PKMζ inhibitor ZIP, Scr-ZIP or protein kinase Cs (PKCs) inhibitor NPC-15437, altered behaviour and spinal PKMζ/PKCs expression were observed. A long-lasting hypersensitivity induced by Car-priming was identified and precipitated by subsequent plantar incision in this 'two-hit' paradigm. Post-treatment with ZIP, but not Scr-ZIP and NPC-15437, after the resolution of Car-priming selectively relieved hypersensitivity. In contrast, pre-priming NPC-15437 treatment only prevented Car-induced initial transient hyperalgesia. Immunoassays showed a significant decrease in spinal PKMζ expression after plantar incision with post-priming ZIP treatment as compared with Scr-ZIP and NPC-15437, but no notable differences in PKCs expression were observed. Spinal PKCs solely contribute to the initial induction of persistent pain, whereas PKMζ plays an essential role in spinal plasticity storage. PKMζ is responsible for the maintenance of peripheral inflammation-primed PPSP. Therefore, spinal PKMζ may be a therapeutic target to prevent surgery-induced chronic pain in patients with preoperative pain.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1β or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1β or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.

The DAPK family: a structure–function analysis

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain. ZIPK's activity is independent of CaM but can be activated by DAPK. The three kinases share some common functions and substrates, such as induction of autophagy and phosphorylation of myosin regulatory light chain leading to membrane blebbing. Furthermore, all can function as tumor suppressors. However, they also each possess unique functions and intracellular localizations, which may arise from the divergence in structure in their respective C-termini. In this review we will introduce the DAPK family, and present a structure/function analysis for each individual member, and for the family as a whole. Emphasis will be placed on the various domains, and how they mediate interactions with additional proteins and/or regulation of kinase function.

Interaction of RhoD and ZIP kinase modulates actin filament assembly and focal adhesion dynamics

RhoD is a member of the classical Rho GTPases and it has an essential role in the regulation of actin dynamics. Furthermore, RhoD also localizes to early endosomes and recycling endosomes, indicating additional roles in the regulation of endosome trafficking. A yeast two-hybrid screen identified Zipper-Interacting Protein Kinase (ZIPK) as a RhoD target. We found that RhoD interacts with ZIP kinase in a GTP dependent manner and modulates actin and focal adhesion reorganization. Interestingly, while ectopic expression of ZIPK in fibroblasts induces actin reorganization and actomyosin contraction seen as stress fiber bundling and membrane blebbing, the concomitant expression of active RhoD suppressed this phenotype. Previously, RhoD has been associated with focal adhesion regulation, and in line with this notion, we observed that ZIPK resulted in reorganization of focal adhesion and increased adhesion size. Importantly, the RhoD activity suppressed ZIPK-dependent effects on FAK activity, indicating a functional interplay between RhoD and FAK in the focal adhesion dynamics.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Rationale: Alterations to the cardiac regulatory light chain (RLC) phosphorylation state correlate with many types of cardiac disease including familial hypertrophic cardiomyopathies (FHC's) and heart failure (HF) post myocardial infarction (MI). However, little is known regarding the role of RLC phosphorylation in regulating cardiac contraction mechanics during shortening. Objective: Determine the mechanical effect of altered cRLC phosphorylation on the force-velocity characteristics of cardiac muscle. Methods and Results: SDS-PAGE electrophoresis determined the effects of heart failure on cRLC phosphorylation on human heart failure (HF) samples and a rat chronic-MI model. We report an increase in RLC phosphorylation in HF progression. Recombinant cRLC was cloned and expressed. Recombinant RLC was differentially phosphorylated in-vitro using cardiac myosin light chain kinase (cMLCK) and ZIP kinase. Recombinant RLC species were exchanged into permeabilised cardiac trabeculae to perform mechanical measurements during shortening (slack test and force-velocity experimentation protocols). We observed a decrease in peak power, velocity at peak power (VPP) and peak isometric force of contraction when reducing RLC phosphorylation. Conversely, enriching RLC phosphorylation increased peak power, VPP and peak maximal unloaded shortening velocity (VMAX) in comparison to control. Conclusions: Altering cRLC phosphorylation has a significant impact on the mechanical function of cardiac muscle and is dramatically altered during HF progression. These results highlight the importance of understanding thick filament regulation and specifically the ability of cRLC phosphorylation to regulate cardiac muscle function.

2012 Late-Breaking Basic Science Oral Session

### Cardiac Myosin Binding Protein C is an Ultra-early and Cardiac-specific Biomarker of Myocardial Necrosis Diederik W Kuster1, Adriana Cardenas-Ospina2, Lawson Miller2, Christian Troidl3, Holger M Nef3, Christoph Liebetrau3, Mollmann Helge3, Karen S Pieper4, Kenneth W Mahaffey4, Neal S Kleiman5, Bruno D Stuyvers2, Ali J Marian6, Sakthivel Sadayappan1; 1Loyola Univ Chicago, Maywood, IL, 2Memorial Univ, St John’s, Canada, 3Kerckhoff Heart and Thorax Cntr, Bad Neuheim, Germany, 4Duke Univ Med Cntr, Durham, NC, 5The Methodist DeBakey Heart and Vascular Cntr, Houston, TX, 6Univ of Texas Health Sciences, Houston, TX Rationale: We recently demonstrated that proteolytic cleavage fragments of cardiac myosin binding protein-C (cMyBP-C) can be detected in the serum after myocardial infarction (MI) in a rat model and in patients with MI. The findings implied that serum cMyBP-C might be useful as cardiac-specific biomarker for MI. The release kinetics of cMyBP-C in the circulation post-MI remains to be elucidated. Objective: Determine the release kinetics of cMyBP-C as an ultra-early and cardiac-specific biomarker of myocardial necrosis. Method and Results: To determine the exact timing of cMyBP-C release in the bloodstream post-acute MI, left anterior descending (LAD) coronary artery was ligated in adult swine (n=6). ECG showed significant ST elevation. Infarct size represented 12.4 ± 1.9% of total ventricular mass. Blood samples were collected before and at predetermined time points between 30 min and 14 days after LAD ligation. Plasma cMyBP-C level was quantified using a highly sensitive and rapid sandwich enzyme-linked immunosorbent assay. Compared with baseline, cMyBP-C levels were increased in post-MI serum within 45 min (0.64 ± 0.52 ng/ml) after LAD ligation and declined after 16 hrs to the baseline level (0.01 ± 0.00 ng/ml). In contrast, cardiac troponin I (cTnI) level peaked after …

Agonist‐Induced Rho Kinase and ZIP kinase Activity Levels in Different Regions of the Stomach

The molecular events that regulate the tonic and phasic phenotypes of smooth muscle are attributed to differential expression of contractile proteins and signaling pathway that regulate MLC kinase and MLC phosphatase activity, and thus MLC20 phosphorylation. The activity of MLC phosphatase is inhibited by Rho kinase/ZIP kinase-dependent pathways. AIM To determine whether Rho kinase and ZIP kinase activity levels correlate with the contractile phenotypes in proximal (tonic) versus distal (phasic) stomach. METHODS Muscle cells were isolated separately from proximal and distal regions of rabbit stomach. Acetylcholine (ACh)-induced stimulation of Rho kinase and ZIP kinase activity was determined by immunokinase assay. RESULTS ACh (0.1 μM) caused stimulation of Rho kinase and ZIP kinase activity in muscle cells of both proximal and distal stomach. However, stimulation of both kinases was significantly higher in muscle cells from proximal stomach compared to distal stomach. Y27632, a selective blocker of Rho kinase, inhibited stimulation of ZIP kinase activity suggesting that stimulation of ZIP kinase activity is dependent on Rho kinase activity. CONCLUSION Higher activity levels of Rho kinase and ZIP kinase, which normally inhibit MLC phosphatase and increase MLC20 phosphorylation, in proximal stomach correlate with its tonic phenotype compared to the distal stomach that exhibits phasic phenotype.

Regulation of RhoA‐Dependent Sustained Contraction by Caveolin‐1 in Gastric Smooth Muscle

The main membrane proteins of caveolae (caveolin-1, −2 and −3) oligomerize within lipid rich domains of smooth muscle plasma membrane and participate in the regulation of G protein-coupled receptor signaling. AIM To identify the role of caveolin-1 in muscarinic m3-receptor mediated stimulation of Rho kinase and ZIP kinase activity in gastric smooth muscle. METHODS Muscle cells were isolated from rabbit stomach, and from wild type and caveolin-1−/− mice. Carbachol (CCh)-induced stimulation of Rho kinase and ZIP kinase activity was determined by immunokinase assay. Muscle contraction was measured by scanning micrometry. RESULTS CCh (0.1 μM) caused stimulation of both Rho kinase and ZIP kinase activity in gastric muscle cells. Y27632 (1 μM), a selective blocker of Rho kinase activity inhibited stimulation of both Rho kinase and ZIP kinase activity suggesting that Rho kinase is an upstream kinase of ZIP kinase. Disruption of caveolae with methyl β-cyclodextrin or suppression of caveolin-1 with caveolin-1 siRNA attenuated the increase in Rho kinase and ZIP kinase activity in response to CCh. Similarly, Rho kinase activation and sustained contraction in response to CCh were significantly attenuated in muscle cells isolated from caveolin-1−/− mice compared to wild type mice. CONCLUSION In gastric smooth muscle caveolin-1 positively regulates Rho kinase/ZIP kinase-dependent sustained muscle contraction.

Involvement of Death-Associated Protein Kinases In DNA Damage Responses of B-ALL Cells.

AbstractAbstract 3369Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway. Disclosures:No relevant conflicts of interest to declare.

Downregulation of ZIP kinase is associated with tumor invasion, metastasis and poor prognosis in gastric cancer

Deletion of 19p13 is one of the most frequent genetic changes in gastric carcinoma (GC), implying the existence of a tumor suppressor gene (TSG) that plays an important role in GC development. To identify the candidate TSG at 19p, array-comparative genomic hybridization (CGH) was applied to study DNA copy-number changes on chromosomes 3, 5p, 13, 16q and 19. The result showed that gains of 16q21, 19q13.1, 5p15.1 and 3q26.31, and losses of 3p21.32, 3p22.2, 19q13.33 and 19p13.3, were frequently detected by array-CGH. One candidate TSG, ZIP kinase (ZIPK), at 19p13.3 was further characterized by immunohistochemistry using a tissue microarray containing 172 primary GCs. Downregulation of ZIPK was detected in 111/162 informative GCs, which was significantly associated with invasion, metastasis and poorer prognosis of GC. To investigate the association of the downregulation of ZIPK with apoptosis, apoptosis assay (TUNEL) was used to compare the apoptotic index between GCs with normal expression and downregulation of ZIPK. TUNEL assay showed that the apoptotic index in GCs with normal ZIPK expression was significantly higher than that in GCs with downregulation of ZIPK (p < 0.001), indicating that ZIPK plays an important pro-apoptotic role in GC. Taken together, we demonstrated here that ZIPK is a tumor suppresser gene and plays an important role in GC development through its pro-apoptotic function. Downregulation of ZIPK can be used to evaluate tumor invasiveness, metastasis and to predict survival of GC.

Long-term cyclic stretch controls pulmonary endothelial permeability at translational and post-translational levels

We have previously described differential effects of physiologic (5%) and pathologic (18%) cyclic stretch (CS) on agonist-induced pulmonary endothelial permeability. This study examined acute and chronic effects of CS on agonist-induced intracellular signaling and cell morphology in the human lung macro- and microvascular endothelial cell (EC) monolayers. Endothelial permeability was assessed by analysis of morphological changes, parameters of cell contraction and measurements of transendothelial electrical resistance. Exposure of both microvascular and macrovascular EC to 18% CS for 2–96 h increased thrombin-induced permeability and monolayer disruption. Interestingly, the ability to promote thrombin responses was present in EC cultures exposed to 48–96 h of CS even after replating onto non-elastic substrates. In turn, physiologic CS preconditioning (72 h) attenuated thrombin-induced paracellular gap formation and MLC phosphorylation in replated EC cultures. Long-term preconditioning at 18% CS (72 h) increased the content of signaling and contractile proteins including Rho GTPase, MLC, MLC kinase, ZIP kinase, PAR1, caldesmon and HSP27 in the pulmonary microvascular and macrovascular cells. We conclude that short term CS regulates EC permeability via modulation of agonist-induced signaling, whereas long-term CS controls endothelial barrier at both post-translational level and via magnitude-dependent regulation of pulmonary EC phenotype, signaling and contractile protein expression.

Physical and functional interactions between ZIP kinase and UbcH5

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination.

Novel Coactivators of Androgen Receptor: AATF and ZIP Kinase

Novel Coactivators of Androgen Receptor: AATF and ZIP Kinase

ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

Myosin Phosphatase Regulatory Pathways

In many ways, the smooth muscle cell (SMC) has been at the forefront in defining cellular signaling pathways. For example, the initial description of agonist-dependent intracellular calcium ([Ca2+]i) transients was made on vertebrate vascular SMC using aqueorin as the indicator.1 Delineation of nitric oxide (NO) and cGMP signaling was first accomplished in smooth muscle.2,3 And more recently, insights into the general mechanisms of gene transcription that dictate phenotypic traits of cells are being defined by smooth muscle specific gene expression.4,5 It should come as no surprise then that the interactions among many signaling pathways in cells should be explained first in the SMC. Perhaps the reason for the central position of the SMC in defining paradigms in cellular biology is that there are well-defined physiologic end points amenable to measurement (ie, contractile force) and even better described and easily quantified biochemical pathways that underlie said physiologic end points (ie, myosin regulatory light chain phosphorylation). From a historical perspective, the role of protein phosphorylation in smooth muscle contraction as a paradigm for cell signaling follows only that for the control of glycogen metabolism (see 6,7 for reviews). Increases in the levels of cytosolic [Ca2+]i initiate smooth muscle contraction by binding to the universal intracellular Ca2+ receptor protein, calmodulin (CaM), which in turn binds to and activates smooth muscle myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation serine-19 of the regulatory myosin light chain (MLC) thereby increasing cross-bridge cycling and the rate of tension development. Dephosphorylation of MLC is catalyzed by smooth muscle specific myosin light chain phosphatase (MLCP). Decreases in cytosolic [Ca2+]i and MLC dephosphorylation are considered salient events in smooth muscle relaxation. What has become apparent only within the last decade is the fact that …