Dysbindin-1, a Schizophrenia-Related Protein,Functionally Interacts with the DNA- Dependent Protein Kinase Complex in an Isoform-Dependent Manner

DTNBP1 has been recognized as a schizophrenia susceptible gene, and its protein product, dysbindin-1, is down-regulated in the brains of schizophrenic patients. However, little is known about the physiological role of dysbindin-1 in the central nervous system. We hypothesized that disruption of dysbindin-1 with unidentified proteins could contribute to pathogenesis and the symptoms of schizophrenia. GST pull-down from human neuroblastoma lysates showed an association of dysbindin-1 with the DNA-dependent protein kinase (DNA-PK) complex. The DNA-PK complex interacts only with splice isoforms A and B, but not with C. We found that isoforms A and B localized in nucleus, where the kinase complex exist, whereas the isoform C was found exclusively in cytosol. Furthermore, results of phosphorylation assay suggest that the DNA-PK complex phosphorylated dysbindin-1 isoforms A and B in cells. These observations suggest that DNA-PK regulates the dysbindin-1 isoforms A and B by phosphorylation in nucleus. Isoform C does not contain exons from 1 to 6. Since schizophrenia-related single nucleotide polymorphisms (SNPs) occur in these introns between exon 1 and exon 6, we suggest that these SNPs might affect splicing of DTNBP1, which leads to impairment of the functional interaction between dysbindin-1 and DNA-PK in schizophrenic patients.     

Protein Kinase Activities Associated with Actin Cytoskeleton in Xenopus laevis Oocytes and Eggs

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [-³²P]ATP has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [-³²P]ATP in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca²⁺-dependent kinases, as well as MAP, p34^cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

ZmMPK6, a Novel Maize MAP Kinase that Interacts with 14-3-3 Proteins

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.     

Glia Maturation Factor (GMF) Interacts with Arp2/3 Complex in a Nucleotide State-dependent Manner

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μm affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.     

The Pseudomonas syringae Effector HopQ1 Promotes Bacterial Virulence and Interacts with Tomato 14-3-3 Proteins in a Phosphorylation-Dependent Manner

A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1’s subcellular localization and virulence-promoting activities in planta.The ability to detect and mount a defense response against pathogenic microbes is vital for plant survival. Plants rely on both passive and active defenses to ward off microbial pathogens. Physical barriers, such as the cell wall and cuticle, as well as chemical barriers provide a first line of defense against microbial colonization. Unlike animals, plants do not possess a circulating immune system and rely on innate immunity for active defenses against microbial pathogens (Spoel and Dong, 2012). Plants use surface-localized receptors to recognize conserved pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin, resulting in pattern-triggered immunity (PTI; Zipfel et al., 2006). Plants also use primarily intracellular nucleotide-binding domain, Leu-rich repeat containing (NLR) immune receptors to recognize pathogen effectors delivered into host cells during infection (Spoel and Dong, 2012). NLR activation results in effector-triggered immunity (ETI). A signature of ETI is the hypersensitive response (HR), a form of programmed cell death occurring at the site of infection.In order to cause disease and suppress host defense responses, gram-negative bacterial pathogens deliver effector proteins into host cells via the type III secretion system (TTSS). Plant pathogenic bacteria deliver a large number (20–40) of effectors into host cells during infection (Cui et al., 2009). Collectively, effectors are required for bacterial virulence (Lindgren et al., 1986). However, knockouts affecting individual effectors frequently have phenotypes that are subtle, likely due to functional redundancy (Cunnac et al., 2011). Alternatively, individual effectors may play an important role in bacterial survival under conditions that are not typically analyzed in the laboratory or act cooperatively with one another. Progress in understanding individual effectors’ contributions to virulence has been made by generating transgenic plants that express effectors. Multiple effectors have been shown to suppress plant innate immunity and promote bacterial growth when either transiently or stably expressed in plants (Jamir et al., 2004; Guo et al., 2009). Effector expression can also result in avirulent phenotypes when a plant NLR receptor recognizes a cognate effector and mounts an HR. Such an HR phenotype can be used to dissect important effector domains required for plant recognition and enzymatic activity.Elucidating effector targets and enzymatic activity is necessary in order to understand how they act to subvert plant immune responses and can provide elegant insight into biological processes. Significant progress has been made in elucidating the enzymatic activity of a subset of effectors. Some of the most well-characterized effectors come from Pseudomonas syringae pv tomato (Pto), the causal agent of bacterial speck on tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Multiple effectors can suppress immune responses by directly targeting PAMP receptors (AvrPto and AvrPtoB) or by interfering with downstream signaling processes (AvrB, AvrPphB, and HopAI1; Cui et al., 2009, 2010). The HopU1 effector interferes with RNA metabolism (Fu et al., 2007), and the HopI1 effector targets heat-shock proteins in the plant chloroplast (Jelenska et al., 2010).14-3-3s are conserved eukaryotic proteins that bind a diverse set of phosphorylated client proteins, typically at one of three distinct 14-3-3 binding motifs (Bridges and Moorhead, 2005). There are common recognition motifs for 14-3-3 proteins that contain phosphorylated Ser or Thr residues, but binding to nonphosphorylated ligands and to proteins lacking consensus motifs has been reported (Henriksson et al., 2002; Smith et al., 2011). The 14-3-3 mode I consensus motif is RXXpS/pTX and that of mode II is RXXXpS/pTXP, where X can be any amino acid and p indicates the site of phosphorylation (Smith et al., 2011). 14-3-3 proteins can also bind to the extreme C termini of proteins at the RXXpS/pTX-COOH mode III consensus motif (Smith et al., 2011). Interaction with 14-3-3s can regulate protein activity by influencing client subcellular localization, structure, and protein-protein interactions (Bridges and Moorhead, 2005). Recently, the Xanthomonas campestris XopN effector was shown to target tomato 14-3-3 isoforms, which facilitates its interaction with the tomato atypical receptor kinase1 and suppresses PTI (Kim et al., 2009; Taylor et al., 2012). Other 14-3-3s have also been shown to play a role during plant defense responses. The tomato TFT7 14-3-3 interacts with multiple mitogen-activated protein kinases to positively regulate HR induced by ETI (Oh and Martin, 2011). The Arabidopsis 14-3-3 isoform λ interacts with the RPW8.2 powdery mildew receptor and is required for complete RPW8.2-mediated resistance (Yang et al., 2009).In this study, we investigated the function of the Pto HopQ1 (for Hrp outer protein Q [also known as HopQ1-1]) effector in tomato. HopQ1 is an active effector that is transcribed and translocated via the TTSS (Schechter et al., 2004). HopQ1 induces cell death when expressed in Nicotiana benthamiana and therefore contributes to differences in host range in P. syringae pathovars on Nicotiana spp. (Wei et al., 2007; Ferrante et al., 2009). HopQ1 was also reported to slightly enhance disease symptoms (approximately 0.2 log) and bacterial virulence on bean (Phaseolus vulgaris) when expressed from P. syringae pv tabaci (Ferrante et al., 2009). Here, we generated transgenic tomato plants expressing HopQ1 that exhibited enhanced susceptibility to virulent Pto as well as the Pto ΔhrcC mutant. HopQ1-interacting proteins were identified from tomato using coimmunoprecipitations coupled with mass spectrometry. Multiple 14-3-3 proteins were identified. HopQ1 possesses a 14-3-3 binding motif whose Ser residue is phosphorylated in planta and affects its association with the tomato 14-3-3s TFT1 and TFT5. Mutation of HopQ1’s 14-3-3 binding motif affected its ability to promote bacterial virulence. Taken together, these results indicate that phosphorylation and subsequent interaction with tomato 14-3-3 proteins affect HopQ1’s virulence-promoting activities and subcellular localization.     

A Novel and Selective p38 Mitogen-Activated Protein Kinase Inhibitor Attenuates LPS-Induced Neuroinflammation in BV2 Microglia and a Mouse Model

Neuroinflammation is an important pathological feature in neurodegenerative diseases. Accumulating evidence has suggested that neuroinflammation is mainly aggravated by activated microglia, which are macrophage like cells in the central nervous system. Therefore, the inhibition of microglial activation may be considered for treating neuroinflammatory diseases. p38 mitogen-activated protein kinase (MAPK) has been identified as a crucial enzyme with inflammatory roles in several immune cells, and its activation also relates to neuroinflammation. Considering the proinflammatory roles of p38 MAPK, its inhibitors can be potential therapeutic agents for neurodegenerative diseases relating to neuroinflammation initiated by microglia activation. This study was designed to evaluate whether NJK14047, a recently identified novel and selective p38 MAPK inhibitor, could modulate microglia-mediated neuroinflammation by utilizing lipopolysaccharide (LPS)-stimulated BV2 cells and an LPS-injected mice model. Our results showed that NJK14047 markedly reduced the production of nitric oxide and prostaglandin E₂ by downregulating the expression of various proinflammatory mediators such as nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α and interleukin-1β in LPS-induced BV2 microglia. Moreover, NJK14047 significantly reduced microglial activation in the brains of LPS-injected mice. Overall, these results suggest that NJK14047 significantly reduces neuroinflammation in cellular/vivo model and would be a therapeutic candidate for various neuroinflammatory diseases.     

Pyruvate Kinase M2 Expression,but Not Pyruvate Kinase Activity,Is Up-Regulated in a Grade-Specific Manner in Human Glioma

Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose de novo or progressed from lower grade tumors, showed a 3–5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth in vitro and in vivo. These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.     

Identification of cyk, a Cyclin B2 Kinase, as a Novel Calcium/Calmodulin-Dependent Protein Kinase II and Its Role during Xenopus laevis Oocyte Maturation

Recently, we have partially purified and characterized a specific cell cycle-regulated cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step (R. Derua, I. Stevens, E. Waelkens, A. Fernandez, N. Lamb, W. Merlevede, and J. Goris, 1997, Exp. Cell Res. 230, 310-324). In the present paper we describe its purification to homogeneity. We could identify the kinase as a special form of calcium/calmodulin-dependent protein kinase II (CaMKII), consisting of five isoforms with molecular masses ranging from 52 to 83 kDa. At least three of them could be considered as novel. Using an in vivo assay with a synthetic peptide (cyktide), an activation of the kinase was shown at about 50% maturation. Further evidence for this observation came from the injection of the calcium chelator BAPTA and the specific cyk/CaMKII inhibitor AIP. A delay of oocyte maturation of at least 1 h was observed. Besides serine 53, a second cyk phosphorylation site in cyclin B2 was identified as threonine 41. Site-directed mutagenesis of these sites indicated that phosphorylation of these sites in Xenopus cyclin B2 was not required for the hallmark functions of cyclin B2.     

CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is α-actinin. We have shown that the CRP1–α-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The K_d for the CRP1–α-actinin interaction is 1.8 ± 0.3 μM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and α-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that α-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of α-actinin. In reciprocal mapping studies, we showed that α-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH₂-terminal 107 amino acids (aa) of CRP1. To determine whether the α-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH₂-terminal part of CRP1 that contains the α-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with α-actinin may be critical for its role in muscle differentiation.     

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, K_is of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.     

Cross Talk between β-Adrenergic and Bradykinin B2 Receptors Results in Cooperative Regulation of Cyclic AMP Accumulation and Mitogen-Activated Protein Kinase Activity

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.