Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3a^KI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways.     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.     

AMPA受体的内化及其分子机制

AMPA受体的内化不仅仅是结束它们的活性状态,受体的许多重要信号功能是与活性依赖的内化密切相关的。突触功能调节中,存在2种形式AMPA受体的内化：组构性内化和活性依赖的内化。现就AMPA受体内化的分类、过程和意义,以及活性依赖的内化的诱发因素、调节因素和内化后的去向进行综述。     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

The FLS2-Associated Kinase BIK1 Directly Phosphorylates the NADPH Oxidase RbohD to Control Plant Immunity

1. Download : Download high-res image (164KB)
2. Download : Download full-size image

     

Systems Biology of Growth Factor-Induced Receptor Endocytosis

Clathrin-mediated endocytosis sorts for degradation of more than 50 different growth factor receptors capable of relaying growth and differentiation signals by means of their cytoplasm-facing, intrinsic tyrosine kinase activity. The kinetics and alternative routings of receptor endocytosis critically regulate growth factor signaling, which underscores the importance of understanding mechanisms underlying fail-safe operation (robustness) and fidelity of the pathway. Like other robust systems, a layered hub-centric network controls receptor endocytosis. Characteristically, the modular hubs (e.g., AP2–Eps15 and Hrs) contain a membrane-anchoring lipid-binding domain, an ubiquitin-binding module, which recruits ubiquitinylated cargo, and a machinery enabling homo-assembly. Scheduled hub transitions, as well as cascades of Rab family guanosine triphosphatases and membrane bending machineries, define points of commitment to vesicle budding, thereby securing unidirectional trafficking. System's bistability permits stimulation by a growth factor, which oscillates a series of switches based on posttranslational protein modifications (i.e., phosphorylation, ubiquitinylation and neddylation), as well as transient low-affinity/high-avidity protein assemblies. Cbl family ubiquitin ligases, along with a set of phosphotyrosine-binding adaptors (e.g., Grb2), integrate receptor endocytosis into the densely wired networks of signal transduction pathways, which are involved in health and disease.     

原癌蛋白c-Cbl促进受体酪氨酸激酶EphA2的降解

c Cbl最近被证明是泛素 蛋白酶体 (ubiquitin proteasome)通路中的一个新的RINGFinger型泛素连接酶 (ubiquitinligase ,E3) .c Cbl可以介导受体酪氨酸激酶和非受体酪氨酸受体激酶的降解 .利用内源性表达较高EphA2的大肠癌细胞株HCT1 1 6 ,通过转染野生型c Cbl和显性负变异体(dominantnegativemutant)c Cbl 70Z ,探讨c Cbl在EphA2降解中的作用 .结果显示 ,c Cbl可促进磷酸化EphA2的降解 ,EphA2的降解必须依赖其配体ephrin A1的刺激 ;利用蛋白酶体 (proteasome)抑制剂MG1 32可抑制磷酸化的EphA2降解 ,提示EphA2的最终降解部位是在蛋白酶体 .研究的结果提示 ,c Cbl作为泛素连接酶诱导磷酸化后的EphA2在蛋白酶体中降解     

Casein Kinase 2 Dependent Phosphorylation of Neprilysin Regulates Receptor Tyrosine Kinase Signaling to Akt

Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.     

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.A rapid and transient increase in reactive oxygen species (ROS), termed an “oxidative burst,” is often associated with responses to abiotic and biotic stresses and could trigger changes in stomatal aperture or programmed cell death in defense against pathogens (Kwak et al., 2003; Torres and Dangl, 2005). ROS production can occur extracellularly through activities of plasma membrane-resident NADPH oxidases (Kangasjärvi et al., 2005; Torres and Dangl, 2005). In plants, Rboh proteins, which are homologs of mammalian NADPH oxidase 2, were shown to be the predominant mediators of apoplastic ROS production (Torres et al., 1998; Galletti et al., 2008). Respiratory burst oxidase homologs D and F (RbohD and RbohF) were identified by mutation to be the responsible oxidases in Arabidopsis (Arabidopsis thaliana) defense responses (Torres et al., 2002). While most ROS generated in response to avirulent Pseudomonas syringae bacteria and Hyaloperonospora oomycete pathogens depend on RbohD function, the induced cell death response by these pathogens appears to be mostly regulated by RbohF. Cell death provoked upon infection with the necrotizing fungus Alternaria, however, is under the control of RbohD (Pogány et al., 2009). The contribution of NADPH oxidases to plant immunity was also described in barley (Hordeum vulgare) and tobacco (Nicotiana benthamiana), where resistance to powdery mildew fungi and the oomycete Phytophthora infestans, respectively, was dependent on Rboh functions (Yoshioka et al., 2003; Trujillo et al., 2006).An early layer of active plant defense is mediated by pattern recognition receptors, which sense microbes according to conserved constituents, so-called pathogen-associated molecular patterns (PAMPs). These initiate a plethora of defense responses referred to as PAMP-triggered immunity (Boller and Felix, 2009). The Arabidopsis receptor kinase Flagellin Sensitive2 (FLS2) recognizes and physically interacts with flg22, the elicitor-active epitope of bacterial flagellin (Felix et al., 1999; Gomez-Gomez and Boller, 2000; Chinchilla et al., 2006). FLS2 is plasma membrane localized and expressed throughout the plant (Robatzek et al., 2006). FLS2 requires the receptor kinase BRI1-Associated Kinase1 (BAK1), which forms a heteromeric complex upon flg22 binding (Chinchilla et al., 2007). Subsequently, a rapid and transient flg22-stimulated oxidative burst occurs that is dependent on RbohD (Zhang et al., 2007). In addition, flg22 triggers early responses, such as ethylene biosynthesis, activation of mitogen-activated protein (MAP) kinase cascades, and changes in gene expression (Felix et al., 1999; Asai et al., 2002; Zipfel et al., 2004). Late flg22 responses include the accumulation of salicylic acid (SA), callose deposition, and an arrest of seedling growth (Gomez-Gomez et al., 1999; Mischina and Zeier, 2007). This collectively contributes to plant immunity (Zipfel et al., 2004; Melotto et al., 2006).Little is known about the regulatory components of FLS2-activated early flg22 responses and their relevance in plant resistance to pathogens. Here, we investigated flg22-triggered ROS production in Arabidopsis seedlings and have identified ethylene signaling as a critical component of the oxidative burst in response to flg22, partly through promoting the accumulation of FLS2. We further provide evidence that the flg22-triggered oxidative burst is required for resistance to bacterial infection at the point of pathogen entry through stomata.     

Glycoxidised LDL Induced the Upregulation of Axl Receptor Tyrosine Kinase and Its Ligand in Mouse Mesangial Cells

Aim/Hypothesis

Low-density lipoprotein (LDL) is subjected to glycoxidation in diabetes, and a novel signalling mechanism by which glycoxidised LDL functions in glomerular mesangial cells remains to be ascertained.

Methods

We performed gene expression analysis in mouse glomerular mesangial cells treated with LDL modified by glycation and oxidation (GO-LDL, 100 µg/ml) for 48 h by using DNA microarray analysis and quantitative real-time PCR. We examined the GO-LDL-specific changes in gene and protein expression in mesangial cells and glomeruli of type 2 diabetic Zucker diabetic fatty (ZDF) rats.

Results

By microarray profiling, we noted that GO-LDL treatment increased Axl receptor tyrosine kinase (Axl) mRNA expression (∼2.5-fold, p<0.05) compared with normal LDL (N-LDL) treatment in mesangial cells. Treatment with GO-LDL also increased the protein levels of Axl and its ligand Gas6 as measured by Western blotting. These increases were inhibited by neutralising Axl receptor-specific antibody. Silencing Gas6 by siRNA inhibited GO-LDL-induced Axl expression in mesangial cells. Axl and Gas6 protein were also increased in cells cultured in high glucose (30 mM) or methylglyoxal (200 µM). Gas6 treatment increased the expression and secretion of TGF-β1 protein, a key regulator of extracellular matrix expression in the glomeruli of diabetic kidneys. Immunohistochemical analyses of glomeruli from 20-week-old ZDF rats exhibited increased Axl protein expression. Rottlerin, a selective PKC-δ inhibitor, completely blocked Gas6-induced TGF-β1 expression.

Conclusions/Interpretation

These data suggest that LDL modified by glycoxidation may mediate Axl/Gas6 pathway activation, and this mechanism may play a significant role in the pathogenesis of diabetic nephropathy.     

Inhibition of G-protein-coupled Receptor Kinase 2 (GRK2) Triggers the Growth-promoting Mitogen-activated Protein Kinase (MAPK) Pathway

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.     

Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2_S938A, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2_S938A or FLS2_D997A (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.     

G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK^−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.     

Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response. If so, insulin resistance would worsen as the level of circulating insulin increases during compensation, which could contribute to the transition of insulin resistance to more severe disease. Here, we investigated the consequences of excess insulin exposure to insulin receptor (IR) activity. Cells chronically exposed to insulin show a diminished the level of IR tyrosine and serine autophosphorylation below that observed after short-term insulin exposure. The diminished IR response did not originate with IR internalization since IR amounts at the cell membrane were similar after short- and long-term insulin incubation. Förster resonance energy transfer between fluorophores attached to the IR tyrosine kinase (TK) domain showed that a change in the TK domain occurred upon prolonged, but not short-term, insulin exposure. Even though the altered ‘insulin refractory’ IR TK FRET and IR autophosphorylation levels returned to baseline (non-stimulated) levels after wash-out of the original insulin stimulus, subsequent short-term exposure to insulin caused immediate re-establishment of the insulin-refractory levels. This suggests that some cell-based ‘memory’ of chronic hyperinsulinemic exposure acts directly at the IR. An improved understanding of that memory may help define interventions to reset the IR to full insulin responsiveness and impede the progression of insulin resistance to more severe disease states.     

Cell Adhesion Properties and Effects on Receptor-Mediated Insulin Endocytosis Are Independent Properties of pp120, a Substrate of the Insulin Receptor Tyrosine Kinase

pp120 undergoes phosphorylation by the tyrosine kinase of the insulin, not the insulin-like growth factor 1 (IGF-1), receptor. Moreover, pp120 stimulates receptor-mediated insulin, but not IGF-1, endocytosis, suggesting that pp120 phosphorylation underlies its effect on insulin endocytosis. pp120 phosphorylation also underlies its bile acid transport and tumor suppression functions. In addition to depending on the intracellular tail, the cell adhesion property of pp120 depends on Arg⁹⁸ in the N-terminal IgV-like ectoplasmic domain. To investigate whether this domain mediates the effect of pp120 on insulin endocytosis, we mutated Arg⁹⁸ to Ala and examined whether this mutation altered pp120 phosphorylation and its effect on ligand endocytosis in transfected NIH 3T3 cells. This mutation did not modify either pp120 phosphorylation or its effect on receptor-mediated ligand endocytosis. These findings support the hypothesis that stimulation of insulin endocytosis by pp120 is not mediated by Arg⁹⁸ in the N-terminal IgV-like ectoplasmic domain of pp120.