Phosphorylation events during viral infections provide potential therapeutic targets

For many medically relevant viruses, there is now considerable evidence that both viral and cellular kinases play important roles in viral infection. Ultimately, these kinases, and the cellular signaling pathways that they exploit, may serve as therapeutic targets for treating patients. Currently, small molecule inhibitors of kinases are under investigation as therapy for herpes viral infections. Additionally, a number of cellular or host-directed tyrosine kinase inhibitors that have been previously FDA approved for cancer treatment are under study in animal models and clinical trials, as they have shown promise for the treatment of various viral infections as well. This review will highlight the wide range of viral proteins phosphorylated by viral and cellular kinases, and the potential for variability of kinase recognition sites within viral substrates to impact phosphorylation and kinase prediction. Research studying kinase-targeting prophylactic and therapeutic treatments for a number of viral infections will also be discussed.     

Myristoyltransferase and calcineurin: novel molecular therapeutic target for epilepsy

N-myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. These myristoylated proteins in the cell have diverse biological functions such as signal transduction, cellular transformation and oncogensis. Known myristoylated proteins [Src family kinases, the catalytic subunit of cAMP-dependent protein kinase and calcineurin (CaN)] are either protein kinases or a protein phosphatases which modulate various cellular metabolic processes. Myristoylation is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The main objective of this review is to focus on the potential role of NMT and CaN in epileptic brain and its involvement in neuronal apoptosis. The findings on the interaction of NMT and CaN with various signaling molecules in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimer's disease, ischemia and epilepsy.     

Protein tyrosine phosphorylation and protein tyrosine nitration in redox signaling

Reversible phosphorylation of protein tyrosine residues by polypeptide growth factor-receptor protein tyrosine kinases is implicated in the control of fundamental cellular processes including the cell cycle, cell adhesion, and cell survival, as well as cell proliferation and differentiation. During the last decade, it has become apparent that receptor protein tyrosine kinases and the signaling pathways they activate belong to a large signaling network. Such a network can be regulated by various extracellular cues, which include cell adhesion, agonists of G protein-coupled receptors, and oxidants. It is well documented that signaling initiated by receptor protein tyrosine kinases is directly dependent on the intracellular production of oxidants, including reactive oxygen and nitrogen species. Accumulated evidence indicates that the intracellular redox environment plays a major role in the mechanisms underlying the actions of growth factors. Oxidation of cysteine thiols and nitration of tyrosine residues on signaling proteins are described as posttranslational modifications that regulate, positively or negatively, protein tyrosine phosphorylation (PTP). Early observations described the inhibition of PTP activities by oxidants, resulting in increased levels of proteins phosphorylated on tyrosine. Therefore, a redox circuitry involving the increasing production of intracellular oxidants associated with growth-factor stimulation/cell adhesion, oxidative reversible inhibition of protein tyrosine phosphatases, and the activation of protein tyrosine kinases can be delineated.     

Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases]

Platelet membrane glycoproteins such as GPIb and GPIa/IIa play important roles in platelet functional responses. They are the receptors for specific ligands (GPIb for von Willebrand factors, and GPIa/IIa for collagen), and the ligand-receptor interaction is the first step that elicits downstream intracellular activation signals which finally culminate in platelet aggregation. Although a variety of signal transduction pathways may be involved, tyrosine kinases appear to be most closely related to platelet activation mediated by membrane glycoproteins. Since its discovery in early 1980's, protein tyrosine phosphorylation catalyzed by tyrosine kinases has been recognized to play a role in regulating the cell function of various cells. Platelets have several Src family tyrosine kinases with an SH2 domain and an SH3 domain. Syk with two SH2 domains also appears to play an important role in platelet activation, especially in its early phase. The SH2 domain binds to a phosphorylated tyrosine residue of other proteins, and the SH3 domain recognizes proline-rich domains of target proteins, thus providing the anchoring sites for protein-protein interactions. In this article, some of the recent developments in the signal transduction pathways related with tyrosine kinases are introduced. Several signaling molecules involved in GPIb- or GPIa/IIa-mediated platelet activation have been identified. Interestingly, the members participating in these processes are distinct, suggesting a diversity of signal transduction mechanisms.     

Redox-linked signal transduction pathways for protein tyrosine kinase activation

The signaling for activation of protein tyrosine kinases (PTKs) is usually started by binding of ligands to cell-surface receptors. However, recent evidence suggests the presence of ligand binding-independent signaling pathways that are mediated by oxidative stress. Oxidation and reduction of protein cysteine sulfhydryl (SH) groups may work as a molecular switch to start or to stop the signaling. It is known that oxidation of cysteine SH groups on protein tyrosine phosphatases switches off the action of protein tyrosine phosphatases. This event may not, however, signal for initial autophosphorylation of previously unphosphorylated PTKs, whereas it certainly prevents dephosphorylation of once-phosphorylated PTKs. We have suggested new mechanisms for oxidative stress-mediated PTK activation. First, cell-surface glycosylphosphatidylinositol-anchoring proteins and a phosphoglycolipid/cholesterol-enriched membrane microdomain termed a "raft" can be the direct targets of oxidative stress for inducing their clustering through an S-S-bonded or S-X-S-bonded crosslinking of cell-surface proteins and subsequent activation of raft-associating Src family PTKs. Second, intracellular specific cysteine SH groups on PTK proteins can be another target of oxidative stress for inducing a conformational change necessary for initial activation of PTKs. A possible relationship between cell-surface and intracellular events is that the former frequently induces superoxide production as the second messenger for the latter.     

Induction of cellular process elongation in hepatic stellate cells cultured on interstitial collagen gel: intracellular signaling mechanism

Hepatic stellate cells (HSCs), located in the perisinusoidal space of Disse, extend long cellular processes, which surround the hepatic sinusoids. However, after primary culture and following subculture using ordinary polystyrene culture dishes, HSCs lost their cellular processes and exhibited myofibroblast-like phenotypes. HSCs displayed rounded shapes when cultured on Matrigel containing the basement membrane components. On the other hand, HSCs exhibited the elongated cellular processes when cultured on interstitial collagen gel. This process elongation was induced by integrin-binding and the subsequent intracellular signaling pathways including protein kinases, protein phosphatases, PI 3-kinases, small G proteins, and microtubule-associated protein (MAP) subtype, MAP2C, and finally resulted from the reorganization of microtubules. The cellular processes contained vitamin A and matrix metalloproteinase-1. Such an HSC culture system using extracellular matrix components would be useful to study HSC functions in vivo, such as retinoid metabolism and reorganization of extracellular matrix.     

Mechanism and consequences of delta-opioid receptor internalization

G protein-coupled delta-opioid receptors (DORs) participate in opioid-mediated analgesia, and chronic opioid application is well known to produce tolerance, limiting the therapeutic use of these drugs. To control and eventually avoid the underlying adaptive mechanisms, several cellular functions were examined with regard to their roles in tolerance development. Specific interest focused on DOR internalization, and the relevant findings are reviewed here. In general, DOR endocytosis is accomplished by complex interactions of various determinants, each having distinct roles in this process. For instance, DOR activation by certain opioids has been shown to turn on the machinery of endocytosis, whereas other opioids stimulate the receptors but fail to bring about internalization. In addition, receptor phosphorylation by different kinases was commonly found to promote DOR sequestration, but receptor internalization also occurs without their phosphorylation. A central role in DOR endocytosis is referred to the adaptor proteins arrestin-2 and arrestin-3, which bind to receptors and subsequently cause the formation of clathrin-coated pits to trigger dynamin-controlled endocytosis. Distinct sorting proteins, kinases, and phosphatases determine whether internalized DORs are delivered either for proteolytic degradation or for recycling, although the underlying mechanisms are hence not clear. Despite intensive studies, understanding of DOR sequestration, degradation, and recycling becomes increasingly difficult. However, the phenomenon of cellular desensitization is recognized to correspond to the loss of responsiveness as consequence of DOR internalization and degradation. In contrast, DOR endocytosis is also discussed to promote resensitization of cells to opioids by recycling of internalized DORs. Even stimulation of extracellular signal-regulated protein kinases (ERK 1/2) may be accomplished by DOR sequestration. However, opposite findings, as well as the fact that multiple cellular mechanisms underly receptor desensitization, resensitization, and ERK activation, questions whether DOR internalization is essential for these processes. Further investigations in both the cellular mechanism and the consequences of DOR endocytosis might thus reveal new aspects of opioid-controlled functions.     

Regulation of peripheral and central immunity: Understanding the role of Src homology 2 domain-containing tyrosine phosphatases,SHP-1 & SHP-2

Protein tyrosine phosphorylation is a potent post-translational regulatory mechanism necessary for maintaining normal physiological functioning of immune cells and it is under the stringent control of antagonizing actions of Protein tyrosine phosphatases and kinases. Two such important Non-Receptor protein tyrosine phosphatases, SHP-1 and SHP-2, have been found to be expressed in immune cells and reported to be key regulators of immune cell development, functions, and differentiation by modulating the duration and amplitude of the downstream cascade transduced via receptors. They also have been conceded as the immune checkpoints & therapeutic targets and hence, it is important to understand their significance intricately. This review compares the roles of these two important cytoplasmic PTPs, SHP1 & SHP-2 in the regulation of peripheral as well as central immunity.     

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.     

NOD-like receptors (NLRs): bona fide intracellular microbial sensors

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) (nucleotide-binding domain leucine-rich repeat containing) family of proteins has been demonstrated to function as regulators of innate immune response against microbial pathogens. Stimulation of NOD1 and NOD2, two prototypic NLRs, results in the activation of MAPK and NF-kappaB. On the other hand, a different set of NLRs induces caspase-1 activation through the assembly of an inflammasome. This review discusses recent findings regarding the signaling pathways utilized by NLR proteins in the control of caspase-1 and NF-kappaB activation, as well as the nonredundant role of NLRs in pathogen clearance. The review also covers advances regarding the cellular localization of these proteins and the implications this may have on pathogen sensing and signal transduction.     

Mechanisms of cell death in oxidative stress

Reactive oxygen or nitrogen species (ROS/RNS) generated endogenously or in response to environmental stress have long been implicated in tissue injury in the context of a variety of disease states. ROS/RNS can cause cell death by nonphysiological (necrotic) or regulated pathways (apoptotic). The mechanisms by which ROS/RNS cause or regulate apoptosis typically include receptor activation, caspase activation, Bcl-2 family proteins, and mitochondrial dysfunction. Various protein kinase activities, including mitogen-activated protein kinases, protein kinases-B/C, inhibitor-of-I-kappaB kinases, and their corresponding phosphatases modulate the apoptotic program depending on cellular context. Recently, lipid-derived mediators have emerged as potential intermediates in the apoptosis pathway triggered by oxidants. Cell death mechanisms have been studied across a broad spectrum of models of oxidative stress, including H2O2, nitric oxide and derivatives, endotoxin-induced inflammation, photodynamic therapy, ultraviolet-A and ionizing radiations, and cigarette smoke. Additionally ROS generated in the lung and other organs as the result of high oxygen therapy or ischemia/reperfusion can stimulate cell death pathways associated with tissue damage. Cells have evolved numerous survival pathways to counter proapoptotic stimuli, which include activation of stress-related protein responses. Among these, the heme oxygenase-1/carbon monoxide system has emerged as a major intracellular antiapoptotic mechanism.     

Phosphatase regulation of macrophage activation

Macrophages are innate immune cells that play critical roles in tissue homeostasis and the immune response to invading pathogens or tumor cells. A hallmark of macrophages is their “plasticity,” that is, their ability to respond to cues in their local microenvironment and adapt their activation state or phenotype to mount an appropriate response. During the inflammatory response, macrophages may be required to mount a profound anti-bacterial or anti-tumor response, an anti-inflammatory response, an anti-parasitic response, or a wound healing response. To do so, macrophages express cell surface receptors for growth factors, chemokines and cytokines, as well pathogen and danger associated molecular patterns. Downstream of these cell surface receptors, cell signalling cascades are activated and deactivated by reversible and competing activities of lipid and protein kinases and phosphatases. While kinases drive the activation of cell signalling pathways critical for macrophage activation, the strength and duration of the signalling is regulated by phosphatases. Hence, gene knockout mouse models have revealed critical roles for lipid and protein phosphatases in macrophage activation. Herein, we describe our current understanding and the key roles of specific cellular phosphatases in the regulation of the quality of macrophage polarization as well as the quantity of cytokines produced by activated macrophages.     

Host factors mediating HIV-1 replication

Human immunodeficiency virus type 1(HIV-1) infection is the leading cause of death worldwide in adults attributable to infectious diseases. Although the majority of infections are in sub-Saharan Africa and Southeast Asia, HIV-1 is also a major health concern in most countries throughout the globe. While current antiretroviral treatments are generally effective, particularly in combination therapy, limitations exist due to drug resistance occurring among the drug classes. Traditionally, HIV-1 drugs have targeted viral proteins, which are mutable targets. As cellular genes mutate relatively infrequently, host proteins may prove to be more durable targets than viral proteins. HIV-1 replication is dependent upon cellular proteins that perform essential roles during the viral life cycle. Maraviroc is the first FDA-approved antiretroviral drug to target a cellular factor, HIV-1 coreceptor CCR5, and serves to intercept viral–host protein–protein interactions mediating entry. Recent large-scale siRNA and shRNA screens have revealed over 1000 candidate host factors that potentially support HIV-1 replication, and have implicated new pathways in the viral life cycle. These host proteins and cellular pathways may represent important targets for future therapeutic discoveries. This review discusses critical cellular factors that facilitate the successive steps in HIV-1 replication.     

间隙连接及其磷酸化调节

间隙连接细胞间通讯(gap junction intercellu lar commun ication,G JIC)是细胞对自身的代谢、增殖、分化等生理过程进行调节的一种重要途径。G JIC的基本组成单位间隙连接蛋白的磷酸化调节是影响G JIC功能的重要因素。蛋白激酶和蛋白磷酸酶通过使间隙连接蛋白磷酸化或脱磷酸化来调节G JIC功能。间隙连接蛋白的适度磷酸化促进G JIC功能的发挥,脱磷酸化或过磷酸化都可抑制G JIC功能,进而影响细胞的正常生理功能。     

The actin-myosin regulatory MRCK kinases: regulation,biological functions and associations with human cancer

The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer.