Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.     

黑色素瘤相关抗原MAAT1p15与LRP6的相互作用及其对Wnt信号通路的调控

为了深入研究Wnt信号的传导机制 ,利用GAL4酵母双杂交系统 ,以Wnt受体LRP6的胞内区为诱饵蛋白 ,筛选小鼠 11 5d胚胎cDNA文库 ,发现了一个新的LRP6相互作用蛋白 :黑色素瘤相关抗原MAAT1p15 (melanoma associatedantigenrecognizedbycytotoxicTlymphocytesp15 ) .免疫共沉淀方法证明了LRP6胞内区和MAAT1p15在哺乳动物细胞中也存在相互作用 .荧光素酶报告系统分析实验显示 ,MAAT1p15能够明显增强Wnt1和LRP6响应的下游基因的转录活性 ,提示MAAT1p15可能是LRP6的一个辅助蛋白     

Actin-binding Protein-1 Interacts with WASp-interacting Protein to Regulate Growth Factor-induced Dorsal Ruffle Formation

Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor–induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor–induced dorsal ruffle formation through its interaction with WIP.     

BMP-1及BMP家族在背-腹轴图式形成中的作用

骨形态发生蛋白（bone morphogenetic proteins, BMPs）是一类在发育过程中起重要作用的分子。除BMP-1外,其他BMP分子均属于转化生长因子-β（transforming growth factor-β, TGF-β）/BMP超家族的发育信号分子。在胚胎发育过程中,这些信号分子通过形成浓度梯度对背—腹轴各向异性分化进行调控。它们借助细胞表面受体的识别进行信号传导,参与调控细胞分化、增殖等活动。而BMP-1则属于细胞外基质金属蛋白酶超家族中的Tolloid蛋白酶家族。BMP-1通过水解其他BMP的抑制物（如脊索发生素,Chordin）,达到促进其他BMP信号传导的目的。BMP-1、BMP和Chordin三者通过相互制约与相互促进等一系列作用,在背—腹沿线建立起稳定的BMP信号梯度。本文就BMP浓度梯度的形成及其稳态维持的机制进行回顾与总结。并在此基础上,对各个物种间BMP浓度梯度形成机制的异同,以及可能存在的协同进化进行比较、分析和讨论。     

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123–510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.Axons of the adult mammalian central nervous system possess an extremely limited ability to regenerate after injury, largely because of inhibitory components of myelin preventing axon growth (1, 2). Several myelin-associated inhibitors have been identified, including myelin-associated glycoprotein (3–5), chondroitin sulfate proteoglycans (6), oligodendrocyte myelin glycoprotein (7), and Nogo (8–10). Myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and Nogo bind to the Nogo-66 receptor (NgR)3 and exert their actions through a tripartite receptor complex NgR/LINGO-1/p75^NTR (11) or NgR/LINGO-1/TROY (12, 13).LINGO-1 is a transmembrane protein that contains a leucine-rich repeat, an immunoglobulin domain, and a short intracellular tail (11). LINGO-1 functions as an essential component of the NgR complexes that mediate the activity of myelin inhibitors to regulate central nervous system axon growth (11, 14). In neurons, the NgR complexes activate RhoA in the presence of myelin inhibitors, which lead to growth cone collapse and neurite extension inhibition (11). Attenuation of LINGO-1 function is able to overcome the myelin inhibitory activity in the spinal cord that prevents axonal regeneration after lesion in rats (15). Besides, it has been reported that LINGO-1 is also expressed in oligodendrocytes, where it negatively regulates oligodendrocyte differentiation and axon myelination (16). Inhibition of LINGO-1 promotes spinal cord remyelination in an experimental model of autoimmune encephalitis (17). Moreover, inhibition of LINGO-1 has been shown to enhance survival, structure, and function of dopaminergic neurons in Parkinson disease models (18). Although the function of LINGO-1 has been intensively studied, much less is known about its downstream signaling.To gain insight into the mechanisms by which LINGO-1 functions, it is of considerable importance to identify new binding partners of LINGO-1. Therefore, using the intracellular domain of LINGO-1 as bait, we employed yeast two-hybrid screening on a brain cDNA library and identified several candidates that interact with LINGO-1, one of which is the protein kinase WNK1.WNKs (with no lysine [K]) are a distinct subfamily of serine-threonine kinases, which are characterized by a unique placement of the lysine that is involved in binding ATP and catalyzing phosphoryl transfer (19). Thus far, WNKs are known composed of four members, WNK1, WNK2, WNK3, and WNK4. Mutations in the serine-threonine kinases WNK1 and WNK4 cause a Mendelian disease PAHII, featuring hypertension and hyperkalemia (20, 21), and their roles in the regulation of electrolyte flux in the kidney have been well established (22). Recently, other important features of WNKs are beginning to be understood. WNKs have also been proposed functioning in a number of non-transport processes, including cell growth, differentiation, and apoptosis (23–26). Although WNK1 has been shown to be expressed in brain (27, 28), little is known about its function in the nervous system until recently; mutations of a nervous system-specific exon of the WNK1 gene were found to cause Hereditary sensory and autonomic neuropathy type II (HSANII) (29). In this study WNK1 was demonstrated to interact with LINGO-1 and regulate Nogo-induced inhibition of neurite extension.     

Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are an important signal for various physiological activities. The Na⁺/Ca²⁺ exchangers (NCX) at the plasma membrane transport Ca²⁺ into or out of the cell according to the electrochemical gradients of Na⁺ and Ca²⁺ to modulate [Ca²⁺]_i homeostasis. Calmodulin (CaM) senses [Ca²⁺]_i changes and relays Ca²⁺ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca²⁺ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM₁₂₃₄ (a Ca²⁺-binding deficient mutant) in HEK293T cells and measured the increase in [Ca²⁺]_i contributed by the influx of Ca²⁺ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM₁₂₃₄. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3^rd or 4^th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2^nd and 3^rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca²⁺]_i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.     

UVR8 Interacts with BES1 and BIM1 to Regulate Transcription and Photomorphogenesis in Arabidopsis

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Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.     

GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability

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The Microtubule-Associated Rho Activating Factor GEF-H1 Interacts with Exocyst Complex to Regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.     

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.     

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.