Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation.

Mammalian cells acquire fatty acids (FAs) from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry-compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.

Cooperative involvement of zipper-interacting protein kinase (ZIPK) and the dual-specificity cell-division cycle 14A phosphatase (CDC14A) in vascular smooth muscle cell migration.

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal adhesion assembly dynamics. ZIPK silencing can induce cytoskeletal remodeling with disassembly of actin stress fiber networks and coincident loss of focal adhesion kinase (FAK)-pY397 phosphorylation. The link between ZIPK inhibition and FAK phosphorylation is unknown, and critical interactor(s) and regulator(s) are not yet defined. In this study, we further analyzed the ZIPK-FAK relationship in VSMCs. The application of HS38, a selective ZIPK inhibitor, to coronary artery vascular smooth muscle cells (CASMCs) suppressed cell migration, myosin light chain phosphorylation (pT18&pS19) and FAK-pY397 phosphorylation as well. This was associated with the translocation of cytoplasmic FAK to the nucleus. ZIPK inhibition with HS38 was consistently found to suppress the activation of FAK and attenuate the phosphorylation of other focal adhesion protein components (i.e., pCas130, paxillin, ERK). In addition, our study showed a decrease in human cell-division cycle 14A phosphatase (CDC14A) levels with ZIPK-siRNA treatment and increased CDC14A with transient transfection of ZIPK. Proximity ligation assays (PLA) revealed CDC14A localized with ZIPK and FAK. Silencing CDC14A showed an increase of FAK-pY397 phosphorylation. Ultimately, the data presented herein strongly support a regulatory mechanism of FAK in CASMCs by a ZIPK-CDC14A partnership; ZIPK may act as a key signal integrator to control CDC14A and FAK during VSMC migration.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

A highly selective Hsp90 affinity chromatography resin with a cleavable linker.

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers.

BACKGROUND: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined. METHODS: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved. RESULTS: HS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells. CONCLUSIONS: The increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.

Expression of membrane Hsp90 is a molecular signature of T cell activation.

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.

Smoothelin-like 1 protein regulates myosin phosphatase-targeting subunit 1 expression during sexual development and pregnancy.

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30-40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.

TAK1: a potent tumour necrosis factor inhibitor for the treatment of inflammatory diseases.

Aberrant tumour necrosis factor (TNF) signalling is a hallmark of many inflammatory diseases including rheumatoid arthritis (RA), irritable bowel disease and lupus. Maladaptive TNF signalling can lead to hyper active downstream nuclear factor (NF)-κß signalling in turn amplifying a cell's inflammatory response and exacerbating disease. Within the TNF intracellular inflammatory signalling cascade, transforming growth factor-ß-activated kinase 1 (TAK1) has been shown to play a critical role in mediating signal transduction and downstream NF-κß activation. Owing to its role in TNF inflammatory signalling, TAK1 has become a potential therapeutic target for the treatment of inflammatory diseases such as RA. This review highlights the current development of targeting the TNF-TAK1 signalling axis as a novel therapeutic strategy for the treatment of inflammatory diseases.

TAK1 regulates the tumor microenvironment through inflammatory, angiogenetic and apoptotic signaling cascades.

Transforming growth factor beta-activated kinase 1 (TAK1) has been implicated for its role in inflammatory signaling and as an important mediator of cellular apoptosis and necroptosis in various cell types. Our recent discovery of a first-in-class, potent and selective TAK1 inhibitor, takinib, represents a novel pharmacological tool to evaluate TAK1's role in cancer. In this study we evaluated the potential therapeutic capacity of TAK1 inhibition on tumor growth and on tumor microenvironment remodeling. In a screen of 16 cancer cell lines, takinib in combination with tumor necrosis factor (TNF) was found to induce cell death (>20%) in 6 out of 16 cell lines. Furthermore, knocking out of TAK1 in MDA-MB-231 cells dramatically increased their sensitization to TNF-mediated apoptosis. In vivo xenographs of MDA-MB-231 TAK1KO tumors displayed delayed tumor growth and increased overall survival compared to TAK1WT controls. Histological and proteomic analysis of TAK1KO tumors showed altered angiogenic signaling and inflammatory signaling via immune cells. Overall, these findings suggest that the targeting of TAK1 in immune mediated cancers may be a novel therapeutic axis.

The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction.

In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) calponin homology (CH)-domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of beta-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t(1/2)) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the calponin homology (CH)-domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess "substrate trapping" properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC(20) in vitro, truncated SMTNL1 lacking the CH-domain was ineffective. In summary, our findings suggest an important role for the CH-domain in mediating the effects of SMTNL1 on smooth muscle contraction.

Smoothelin-like 1 deletion enhances myogenic reactivity of mesenteric arteries with alterations in PKC and myosin phosphatase signaling.

The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40 mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60-120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.

Pharmacological inhibition of TAK1, with the selective inhibitor takinib, alleviates clinical manifestation of arthritis in CIA mice.

OBJECTIVES: To examine the ability of takinib, a selective transforming growth factor beta-activated kinase 1 (TAK1) inhibitor, to reduce the severity of murine type II collagen-induced arthritis (CIA), and to affect function of synovial cells. METHODS: Following the induction of CIA, mice were treated daily with takinib (50 mg/kg) and clinical scores assessed. Thirty-six days post-CIA induction, histology was performed on various joints of treated and vehicle-treated animals. Inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation were quantified. Furthermore, pharmacokinetics of takinib were evaluated by LC-MS in various tissues. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells were cultured with 10 µM takinib and cytokine secretion analyzed by cytokine/chemokine proteome array. Cytotoxicity of takinib for RA-FLS was measured with 24 to 48 h cultures in the presence or absence of tumor necrosis factor (TNF). RESULTS: Here, we show takinib's ability to reduce the clinical score in the CIA mouse model of rheumatoid arthritis (RA) (p < 0.001). TAK1 inhibition reduced inflammation (p < 0.01), cartilage damage (p < 0.01), pannus, bone resorption, and periosteal bone formation and periosteal bone width in all joints of treated mice compared to vehicle treated. Significant reduction of inflammation (p < 0.004) and cartilage damage (p < 0.004) were observed in the knees of diseased treated animals, with moderate reduction seen in the forepaws and hind paws. Furthermore, the pharmacokinetics of takinib show rapid plasma clearance (t½ = 21 min). In stimulated RA-FLS cells, takinib reduced GROα, G-CSF, and ICAM-1 pro-inflammatory cytokine signaling. CONCLUSION: Our findings support the hypothesis that TAK1 targeted therapy represents a novel therapeutic axis to treat RA and other inflammatory diseases.

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion.

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

Molecular biologist's guide to proteomics.

The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems.

Dual kinase-mediated regulation of PITK by CaMKII and GSK3.

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIdelta), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.