Molecule Kinase Research Articles

Effects of the Rho kinase inhibitor, hydroxyfasudil, on bladder dysfunction and inflammation in rats with HCl‐induced cystitis

To evaluate the effect of the Rho kinase inhibitor, hydroxyfasudil, on bladder function in a rat model of HCl-induced chemical cystitis, and to elucidate the possible mechanisms associated with its therapeutic effect. Female Sprague-Dawley rats with HCl-induced cystitis were given hydroxyfasudil (10 mg/kg, i.p.) for 7 days. Treatment efficacy was determined by comparing bladder function and histopathology to sham and untreated control rats. Bladder function was determined by cystometric analysis. Rho kinase activity was determined by quantitative reverse transcription polymerase chain reaction and signal inhibition of downstream Ras homolog member A/Rho kinase signaling molecules by western blot and immunohistochemistry. Treatment with hydroxyfasudil significantly improved bladder intercontraction intervals. Rats treated with hydroxyfasudil also showed a significant reduction of histopathological features associated with cystitis. Western blot and immunohistochemistry findings showed that hydroxyfasudil inhibited downstream molecules of Rho kinase that ameliorated changes associated with HCl-induced chemical cystitis, such as inflammatory cell recruitment and smooth muscle cell proliferation. The findings from the present study suggest a promising therapeutic role for hydroxyfasudil in bladder inflammation associated with cystitis.

The Tomato Prf Complex Is a Molecular Trap for Bacterial Effectors Based on Pto Transphosphorylation

The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery. AvrPto and AvrPtoB are two such effectors from Pseudomonas syringae, which disable an overlapping range of kinases in Arabidopsis and Tomato. Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop. AvrPto is an inhibitor of Pto kinase activity, but paradoxically, this kinase activity is a prerequisite for defense activation by AvrPto. Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Both P+1 loop disruption and transphosphorylation are necessary for signalling. Thus, effector perturbation of one kinase molecule in the complex activates another. Hence, the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases, an essential aspect of the pathogen's virulence strategy. The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen.

Imaging of Fluorescent ATP-Stabilized Smooth Muscle Myosin Filaments, Actin Filaments/Bundles and Single Molecules of Myosin Light Chain Kinase (MLCK) under Conditions of SMM Phosphorylation

We are using fluorescence microscopy to characterize the interaction of single active MLCK molecules with smooth muscle myosin (SMM) filaments and acto-SMM bundles in an in vitro¬ model system to determine the mechanism by which MLCK phosphorylates SMM. Since ATP is required for the SMM ATPase and the MLCK kinase activity, this requires unphosphorylated SMM filaments that are stable in the presence of ATP, a condition that normally causes disassembly. We have prepared and characterized modified SMM filaments for this study. They are 1) covalently labeled with NHS-Rhodamine for ease of imaging, 2) lightly cross-linked with EDC on the heavy chain so they are stable to ATP-induced disassembly, 3) similar to native SMM filaments in length, 4) fully regulated by phosphorylation measured by acto-SMM ATPase activity, and the ability to move actin in an in vitro motility assay, and 5) able to dissociate from actin in an [ATP]-dependent manner. Interestingly, these modified SMM filaments are capable of bundling actin filaments in the presence of ATP, calmodulin (CaM), and Ca2+; conditions that are sufficient for MLCK to phosphorylate SMM. SMM filaments preferentially bind to actin bundles rather than single actin filaments, and as ATP is titrated into a preparation of acto-SMM bundles, they dissociate leaving short actin filaments which are predominantly bound to SMM filaments. We have imaged the interaction of single quantum dot-labeled MLCK (QD-MLCK) molecules with these SMM filaments and acto-SMM bundles using total internal reflection fluorescence (TIRF) microscopy to visualize single QD-MLCK. We observed QD-MLCK colocalizing with SMM filaments and acto-SMM bundles and will characterize the QD-MLCK interaction with both proteins in the presence of ATP before and after activation of MLCK by Ca2+-CaM.

Effects of Bariatric Surgery on Adipokine-Induced Inflammation and Insulin Resistance

Over a third of the US population is obese and at high risk for developing type 2 diabetes, insulin resistance, and other metabolic disorders. Obesity is considered a chronic low-grade inflammatory condition that is primarily attributed to expansion and inflammation of adipose tissues. Indeed, adipocytes produce and secrete numerous proinflammatory and anti-inflammatory cytokines known as adipokines. When the balance of these adipokines is shifted toward higher production of proinflammatory factors, local inflammation within adipose tissues and subsequently systemic inflammation occur. These adipokines including leptin, visfatin, resistin, apelin, vaspin, and retinol binding protein-4 can regulate inflammatory responses and contribute to the pathogenesis of diabetes. These effects are mediated by key inflammatory signaling molecules including activated serine kinases such as c-Jun N-terminal kinase and serine kinases inhibitor κB kinase and insulin signaling molecules including insulin receptor substrates, protein kinase B (PKB, also known as Akt), and nuclear factor kappa B. Bariatric surgery can decrease body weight and improve insulin resistance in morbidly obese subjects. However, despite reports suggesting reduced inflammation and weight-independent effects of bariatric surgery on glucose metabolism, mechanisms behind such improvements are not yet well understood. This review article focuses on some of these novel adipokines and discusses their changes after bariatric surgery and their relationship to insulin resistance, fat mass, inflammation, and glucose homeostasis.

Neoechinulin A suppresses amyloid-β oligomer-induced microglia activation and thereby protects PC-12 cells from inflammation-mediated toxicity

A pathological hallmark of Alzheimer's disease (AD), aggregation and deposition of amyloid-β peptides, has been recognized as a potent activator of microglia-mediated neuroinflammation and neuronal dysfunction. Therefore, downregulation of microglial activation has a significant therapeutic demand. In this study, focus was given to evaluate the ability of neoechinulin A, an indole alkaloid isolated from marine-derived Microsporum sp., to attenuate microglial activation by oligomeric amyloid-β 1–42 (Aβ42). Neoechinulin A treatment significantly inhibited the generation of reactive oxygen and nitrogen species in Aβ42-activated BV-2 microglia cells. In addition, we found that neoechinulin A significantly suppressed the production of neurotoxic inflammatory mediator tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in activated BV-2 cells. Moreover, the treatment downregulated the protein and gene expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β and IL-6. Further, activated microglia-mediated apoptosis of PC-12 pheochromocytoma cells was significantly repressed by neoechinulin A. The molecular mechanism studies suggested that neoechinulin A may block the phosphorylation of mitogen-activated protein kinase (MAPK) molecule p38, apoptosis signal-regulating kinase 1 (ASK-1) and nuclear translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits. Regulation of these signalling pathways have most probably contributed to the anti-inflammatory activity of neoechinulin A. Collectively, these results suggest that with further studies neoechinulin A have a potential to be developed as a modulator of neuroinflammatory process in AD.

DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, inhibiting cell migration and invasion

The aim of this study was to explore the molecular mechanisms of the diallyl disulfide (DADS)-mediated downregulation of LIM kinase-1 (LIMK1) and the consequent inhibition of the migration and invasion of human colorectal cancer cells. RNA interference technology was used to establish stable LIMK1-miRNA/SW480 cell lines. The effects of DADS and LIMK1 RNA interference on the migration and invasion of SW480 cells were observed by scratch wound healing assay and Transwell migration assay. The effects of DADS on signaling molecules of the Rac1-Rho kinase (ROCK)1/p21-activated kinase (PAK)1-LIM kinase (LIMK)1-actin depolymerizing factor (ADF)/cofilin pathway in SW480 cells were examined by RT-PCR and western blot analysis. The healing and migration rate of the SW480 cells was significantly reduced and the cell penetrating ability was significantly suppressed (P<0.05) following treatment with DADS (45mg/l). The immunohistochemistry and western blot analysis results showed that DADS significantly downregulated LIMK1 protein expression and suppressed LIMK1 protein phosphorylation. Furthermore, the RT-PCR and western blot analysis results revealed that DADS suppressed Rac1, ROCK1, PAK1, LIMK1 and destrin mRNA and protein expression, as well as the protein phosphorylation of LIMK1 and cofilin1. The data demonstrate that LIMK1 expression positively correlates with the SW480 cell migration and invasion ability. DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, suppressing SW480 cell migration and invasion.

Differential expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in orange-spotted grouper (Epinephelus coioides)

Nucleotide-binding oligomerization domain-containing proteins-1 and -2 (NOD1 and NOD2) are members of the NOD-like receptors (NLRs) family. They are both cytoplasmic receptors, and sense microbial infections/danger molecules to induce host innate immune response. In this study, the full-length ORF sequences of NOD1 and NOD2 were cloned, and the putative amino acid sequences were identified in orange-spotted grouper (Epinephelus coioides). The complete open reading frame (ORF) of grouper NOD1 contained 2823 bp encoding a 940 amino acid protein. Grouper NOD2 cDNA contained a 2967 bp ORF, encoding a protein of 988 amino acid residues. Both grouper NOD1 and NOD2 had similar domains to human and fish counterparts. Phylogenetic tree analysis showed that grouper NOD1 clustered with grass carp, zebrafish and channel catfish, while NOD2 was most closely related to fugu. Expression patterns of grouper NOD1 and NOD2 were next studied. NOD1 had the highest level of expression in skin while NOD2 in trunk kidney. Post Vibrio alginolyticus (strain EcGS020401), lipopolysaccharide (LPS) or PolyI:C challenges, gene expression of grouper NOD1 and NOD2 was stimulated to different extents. NOD1 showed a significant enhancement after LPS stimulation, but NOD2 increased more significantly after PolyI:C invasion, indicating that NOD1 and NOD2 may exert different effects on the eradication of bacteria and virus. The adaptor protein RIP-like-interacting CLARP kinase (RICK) and downstream molecule interleukin-8 (IL-8) were also induced at different levels after stimulation, which indicated that NOD1 and NOD2 signal transduction was involved in grouper innate immune protection against bacterial and viral infections.

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

Abstract 5755: PR/AMPK-IGF/PI3K network plays a major role on progestin-resistance in endometrial cancer therapy

Abstract Progestin-resistance is a main obstacle to the success of conservative therapy in type I young endometrial cancer patients. We have established a progestin-resistant endometrial cancer cell line by long-term induction with gradual increasing of progestin concentration. Our previous study confirmed that progestin activated PI3K/Akt pathway through a non-genomic pathway independent of Progesterone receptor (PR). Blocking PI3K pathway up-regulated PR expression and restored the anti-proliferative effect of medoxyprogesterone acetate (MPA) in progestin-resistant cells. We propose that PI3K/Akt activation may not be the only mechanism of progestin-resistance. To further explore other molecular events leading to progestin-resistance and to search for any possible approaches to reverse this resistance in endometrial cancer, we have characterized functional proteomics by reverse phase protein array (RPPA) in progestin-sensitive and -resistant endometrial cancer cells, and the RPPA results were confirmed by Western blots. Our results demonstrated up-regulation of Insulin-like growth factor-1 receptor (IGF-1R), and down-regulation of IGF binding protein 2 (IGFBP2) and Ataxia Telangiectasia (ATM) [an upstream molecule of adenosine monophosphate-activated kinase (AMPK) pathway] in progestin-resistant cells, suggesting that IGF/AMPK metabolic pathways are involved in progestin-resistance of endometrial cancer therapy. We further identified the differential effects of insulin stimulation on metabolic changes in progestin-sensitive and -resistant cell lines. We found that acute stimulation with high concentration of insulin not only activated PI3K/Akt, but also reduced the expression of PR, EGFR and IGF-1R, and further mediated proliferation of primary endometrial cancer cells. Continually stimulation with insulin reduced MPA-mediated anti-proliferation effect in progestin-sensitive cells, but no effect in progestin-resistant cells, indicating the existing of altered insulin-responsiveness in progestin-resistant cells. Furthermore, Metformin, an insulin-independent AMPK activator and is commonly used as an insulin sensitizer, could restore progestin sensitivity in progestin-resistant endometrial cancer cells. The restoration was demonstrated directly related to the inhibition of PI3K/Akt pathway, activation of AMPK and upregulation of PR transcription. In summary, our study revealed that PR/AMPK-IGF/PI3K network plays an important role on progestin-resistance in endometrial cancer therapy. The combinatory treatment of progestin with PI3K inhibitor, or with AMPK activator-metformin might be an effective conservative approach for endometrial cancer patients who developed progestin-resistance in their therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5755. doi:1538-7445.AM2012-5755

A Common Model for Cytokine Receptor Activation: Combined Scissor-Like Rotation and Self-Rotation of Receptor Dimer Induced by Class I Cytokine

The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin) were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.

PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia

Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.

Stable Complexes of LYN Kinase and Druglike Small Molecules

Glioblastoma multiforme (GBM) is a very aggressive brain cancer in humans. Even with improvements in surgical, radiation, and chemotherapy treatments, current prognosis is bleak, with only a 14-month median survival time. LYN, an important kinase involved in cell regulation, is often overexpressed in GBM. This work addresses computational design of druglike small molecules that could potentially inhibit LYN and thus hinder GBM progression. New putative LYN inhibitors were obtained by atomic substitutions and structural alterations of bafetinib, a small molecule previously found to bind LYN. Druglike properties and toxicities of the designed molecules were evaluated using the Osiris Property Explorer program. Molecules with no implied toxicities and most favorable druglike properties were used for docking studies in the ArgusLab program. Designed molecules that made the most stable docking configurations with LYN, but no stable configurations with other kinases, were identified as LYN-specific. Binding energies of the stable complexes formed by these molecules and LYN were calculated. Possible utilization of the designed molecules in drug research against GBM is discussed.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

A Common Model for Cytokine Receptor Activation: Combined Scissor-Like Rotation and Self-Rotation of Receptor Dimer Induced by Class I Cytokine

The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1:2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1:1 complex. Here we computed the binding pathway leading to the 1:2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin) were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1:1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1:2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a number of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and be useful for elucidating activation mechanisms of other receptors.

Anti-Epidermal Growth Factor/Epidermal Growth Factor Receptor Therapeutic Anti-cancer Drugs and the Wound Healing Process

Cutaneous wound healing is a complex process involving blood clotting, inflammation, tissue formation, and tissue remodeling. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. The use of targeted anti-cancer agents is increasing. It is common to utilize a multi-modal treatment approach towards solid tumors, often including surgical resection, and it has become apparent that some targeted agents can impair wound healing or cause increasing risk of perioperative complications. There are limited data regarding the wound healing process of anti-cancer target drugs blocking the EGF/EGFR system. The aim of this paper is to review and to comment the effects of anti- EGF/EGFR drugs on the skin wound healing process after programmed or emergency surgical procedures. A review of the current literature, including our own results, was undertaken. We included the monoclonal antibodies cetuximab, panitumumab, nimotuzumab; the small tyrosine kinase molecules erlotinib and gefitinib; and the EGFbased cancer vaccine; CIMAvax and the EGFR-based cancer vaccine; HER-1 vaccine. Apparently, there are no deleterious effects of the anti-EGF/EGFR drugs in the wound healing post-operative process. Taking into account that treatment with anti-EGF/EGFR drugs inhibits tumor cell proliferation, and the lack of deleterious effects of these EGF/EGFR specific inhibitors in the wound healing post-operative process; we suggest that these kinds of drugs could be maintained and their effects tested, with very special surveillance during the post-surgical period.

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10-100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925-949 and 1,140-1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.

Virus manipulation of cell cycle

Viruses depend on host cell resources for replication and access to those resources may be limited to a particular phase of the cell cycle. Thus manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment. For example, viruses capable of infecting nondividing cells induce S phase in order to activate the host DNA replication machinery and provide the nucleotide triphosphates necessary for viral DNA replication (Flemington in J Virol 75:4475-4481, 2001; Sullivan and Pipas in Microbiol Mol Biol Rev 66:179-202, 2002). Viruses have developed several strategies to subvert the cell cycle by association with cyclin and cyclin-dependent kinase complexes and molecules that regulate their activity. Viruses tend to act on cellular proteins involved in a network of interactions in a way that minimal protein-protein interactions lead to a major effect. The complex and interactive nature of intracellular signaling pathways controlling cell division affords many opportunities for virus manipulation strategies. Taking the maxim "Set a thief to catch a thief" as a counter strategy, however, provides us with the very same virus evasion strategies as "ready-made tools" for the development of novel antivirus therapeutics. The most obvious are attenuated virus vaccines with critical evasion genes deleted. Similarly, vaccines against viruses causing cancer are now being successfully developed. Finally, as viruses have been playing chess with our cell biology and immune responses for millions of years, the study of their evasion strategies will also undoubtedly reveal new control mechanisms and their corresponding cellular intracellular signaling pathways.

Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity

BackgroundIt has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone.ResultsWe used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator.ConclusionsA proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective manner. We believe our results support the position that such characterizations should consider retroactivity as a robust potential source of off-target effects induced by kinase inhibitors and other targeted therapies.

1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] suppresses pro-inflammatory responses by blocking NF-κB and MAPK signaling pathways in activated microglia

Unregulated activation of microglia is a key risk factor contributes to neurodegenerative diseases and suppression of this phenomenon is considered as a potential therapeutic target. The compound isolated from sea horse Hippocampus kuda Bleeler; 1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] was characterized for its ability in suppressing LPS mediated activation of murine BV-2 cells. Despite the presence of various active molecular groups in the structure, SE1 has not well explored for its biological activities. The outcome of this study clearly indicated that SE1 inhibited the production of inflammatory mediators; nitric oxide, prostaglandin E(2) and pro-inflammatory cytokines. Furthermore, it inhibited the protein and gene expression levels of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β and interleukin-6. The responsible signaling mechanisms leading to these inhibitions were identified as SE1 mediated blocking of phosphorylation of mitogen activate protein kinase (MAPK) molecules; C-jun-N-terminal kinase (JNK), p38 and nuclear translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits. These results suggest that SE1 has the potential to be further developed as therapeutic against neuro-inflammation.

Exact solutions to a spatially extended model of kinase–receptor interaction

B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.