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Yayici Köken, Özlem, Ülkühan Öztoprak, Vehap Topçu, Büsranur Çavdarli, Çagri Mesut Temucin, Üstün Aydingöz, Özge Dedeoglu Toptas, Hulya Kayilioglu, and Deniz Yuksel. "Expanding the genotype-phenotype spectrum of autosomal recessive Charcot-Marie-Tooth disease: A novel PLEKHG5 gene mutation." Neurology Asia 26, no. 3 (September 2021): 607–12. http://dx.doi.org/10.54029/2021jmr.
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Autosomal recessive intermediate Charcot Marie Tooth (CMT) disease type C is a very rarely-seen neurogenetic disorder. Homozygous or compound heterozygous mutation in the Pleckstrin homology domain-containing family G member 5 (PLEKHG5) gene on chromosome 1p36 was recently reported in patients with CMT. From the first description of the disease to date, almost 40 different variants associated with the PLEKHG5 gene were identified. Here, we present an adolescent girl who was thought initially to be myopathy because of progressive proximal muscle weakness. The electrophysiologic study revealed axonal sensory and motor neuropathy with some demyelinating features. She was diagnosed with autosomal recessive inheritance, intermediate CMT disease type C with a novel homozygous mutation in the PLEKHG5 gene in clinical exome sequencing as c.1600- 2A>G by next-generation sequencing. We describe here the novel mutation in the PLEKHG5 gene and the genotype-phenotype correlation.
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AshaRani, P. V., Syidda Amron, Noor Azizah Bte Zainuldin, Sumanty Tohari, Alvin Y. J. Ng, Guo Song, Byrappa Venkatesh, and Ajay S. Mathuru. "Whole-Exome Sequencing to Identify Potential Genetic Risk in Substance Use Disorders: A Pilot Feasibility Study." Journal of Clinical Medicine 10, no. 13 (June 25, 2021): 2810. http://dx.doi.org/10.3390/jcm10132810.
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Genetics intersects with environmental, cultural, and social factors in the development of addictive disorders. This study reports the feasibility of whole-exome sequencing of trios (subject and two family members) to discover potential genetic variants in the development of substance use disorders (SUD). Family trios were recruited from the National Addictions Management Service in Singapore during the 2016–2018 period. Recruited subjects had severe alcohol use disorder (AUD) or opioid use disorder (OUD), with nicotine dependence (ND) and a family history of addictive disorders. Demographic characteristics and severity of addiction were captured. Whole-exome sequencing (WES) and analysis were performed on salivary samples collected from the trios. WES revealed variants in several genes in each individual and disruptive protein mutations in most. Variants were identified in genes previously associated with SUDs, such as Pleckstrin homology domain-containing family M member 3 (PLEKHM3), coiled-coil serine-rich protein 1 (CCSER1), LIM and calponin homology domains-containing protein 1 (LIMCH1), dynein axonemal heavy chain 8 (DNAH8), and the taste receptor type 2 member 38 (TAS2R38) involved in the perception of bitterness. The feasibility study suggests that subjects with a severe addiction profile, polysubstance use, and family history of addiction may often harbor gene variants that may predispose them to SUDs. This study could serve as a model for future precision medicine-based personalized interventional strategies for behavioral addictions and SUDs and for the discovery of potentially pathogenic genetic variants.
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Tellermann, A., T. Witte, C. Lansche, M. Stoll, RE Schmidt, and NT Baerlecken. "Autoantibodies binding to ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) are associated with mycobacterial infections." HIV Medicine 16, no. 2 (September 12, 2014): 114–21. http://dx.doi.org/10.1111/hiv.12194.
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Nowak, Daniel, Norihiko Kawamata, Birte Niebuhr, Verena Nowak, Maximilian Mossner, Rahul R. Nahar, Nils Heinrich Thoennissen, et al. "The Pax5 Fusion Product Pax5-C20orf112 Causes Downregulation of Pre-B Cell Receptor Genes and Induces Differential Proliferation Patterns in B-Lymphoblastic Cell Lines." Blood 114, no. 22 (November 20, 2009): 1284. http://dx.doi.org/10.1182/blood.v114.22.1284.1284.
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Abstract Abstract 1284 Poster Board I-306 Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. Disclosures No relevant conflicts of interest to declare.
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Ren, Xiu-Rong, Quan-Sheng Du, Yang-Zhong Huang, Shi-Zhou Ao, Lin Mei, and Wen-Cheng Xiong. "Regulation of Cdc42 Gtpase by Proline-Rich Tyrosine Kinase 2 Interacting with Psgap, a Novel Pleckstrin Homology and Src Homology 3 Domain Containing Rhogap Protein." Journal of Cell Biology 152, no. 5 (March 5, 2001): 971–84. http://dx.doi.org/10.1083/jcb.152.5.971.
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Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.
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Ma, Alice, and Charles Abrams. "Pleckstrin Homology Domains and Phospholipid-Induced Cytoskeletal Reorganization." Thrombosis and Haemostasis 82, no. 08 (1999): 399–406. http://dx.doi.org/10.1055/s-0037-1615859.
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IntroductionA remarkable event that takes place during platelet activation is the reorganization that occurs when platelets adhere and spread on exposed collagen fibrils or become activated in the circulation by agonists, such as thrombin or adenosine diphosphate (ADP). In response to either stimulus, the shape of the platelet changes from a smooth disc to an irregular form with multiple, finger-like projections. This transformation is due to cytoskeletal rearrangements within the platelet. The platelet cytoskeleton is an intricately woven network1 arranged in three major structures: a cytoplasmic actin network, a rim of membrane-associated cytoskeleton, and a marginal band consisting of a microtubule coil. Together, these lend support to the platelet plasma membrane and give shape to both resting and activated platelets.At several levels, phosphoinositides are involved in the regulation of the platelet cytoskeleton. Actin binding, capping, and severing proteins are regulated by binding to phosphatidylinositol 4,5-bisphosphate (PIP2). The action of specific phosphoinositide kinases and phosphatases, leading to the regulation of levels of D3- and D4-containing phosphoinositides, has a profound impact on actin assembly. For example, synthesis of D3-containing phosphoinositides by phosphoinositide 3-kinases (PI3Ks) can lead to cortical actin assembly and the formation of lamellipodia downstream of stimulation by growth factor receptors, insulin receptors, and G protein-coupled receptors.2-5 There is increasing evidence that other lipid kinases also regulate cytoskeletal reorganization. Phosphatidylinositol 4-P 5-kinase enzymes, acting downstream of Rho family GTPases, have been shown to stimulate actin assembly.6 Because these areas have been covered in other articles,7,8 this review will, instead, concentrate on the role of pleckstrin and pleckstrin homology (PH) domains in the regulation of the actin cytoskeleton.Pleckstrin homology (PH) domains are the most wellrecognized phosphoinositide-binding protein motifs, comprising “modules” within more than 100 signaling proteins, and are used to mediate intermolecular interactions. The threedimensional structures of all PH domains studied to date are virtually superimposable, despite divergence in their amino acid sequence.9-17 The basic PH domain structure is composed of a β “sandwich,” capped at one end by a carboxyl-terminal α-helix, and all PH domains exhibit a striking polarity in their distribution of surface charge (Fig. 1). Based on the similarity of the structure of the NH2-terminal PH domain of pleckstrin to that of the retinol-binding protein, which was known to bind lipids, Harlan and coworkers tested PH domains and demonstrated that they bind to phosphoinositides.18 Since then, a number of laboratories, including our own, have published reports showing that the binding of PH domains to phosphoinositides can regulate protein function.4,19-22 It is now accepted that PH domains serve to localize their molecules into membrane structures by binding to phosphoinositides;18,23 though some PH domains may interact with other targets, such as the βγ subunits of heterotrimeric G proteins (Gβγ)24-27 or protein kinase C (PKC).28-30 The structure of several PH domains complexed to inositol trisphosphate (IP3) has been solved,11,13 confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain. For example, the association of the PH domain of PLCδ with IP3 is shown in Figure 1. Pleckstrin is a 43-kDa hematopoietic protein that contains the amino- and carboxyl- termini of the two prototypic PH domains (Fig. 2). Pleckstrin was first described as a major substrate for PKC in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Though its function in vivo remains unclear, expressed pleckstrin can affect PIP2-based signaling mediated by phospholipase C, PI3K, and inositol phosphatases.31-33 Ser113, Thr114, and Ser117, the three residues phosphorylated by PKC, lie adjacent to, but not within, the amino-terminal PH domain. Phosphorylation at these sites has been shown to regulate the function of this PH domain.34 Recently, a third functional motif has been described within pleckstrin.35 This motif is termed the DEP domain after the first three proteins known to possess this sequence (disheveled, Egl-10, and pleckstrin).
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Del Fattore, Andrea, Rachele Fornari, Liesbeth Van Wesenbeeck, Fenna de Freitas, Jean-Pierre Timmermans, Barbara Peruzzi, Alfredo Cappariello, et al. "A New Heterozygous Mutation (R714C) of the Osteopetrosis Gene, Pleckstrin Homolog Domain Containing Family M (With Run Domain) Member 1 (PLEKHM1), Impairs Vesicular Acidification and Increases TRACP Secretion in Osteoclasts." Journal of Bone and Mineral Research 23, no. 3 (November 12, 2007): 380–91. http://dx.doi.org/10.1359/jbmr.071107.
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Meller, Nahum, M. Jody Westbrook, John D. Shannon, Chittibabu Guda, and Martin A. Schwartz. "Function of the N-terminus of zizimin1: autoinhibition and membrane targeting." Biochemical Journal 409, no. 2 (December 21, 2007): 525–33. http://dx.doi.org/10.1042/bj20071263.
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Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.
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Derrien, Valérie, Carole Couillault, Michel Franco, Stéphanie Martineau, Philippe Montcourrier, Rémi Houlgatte, and Philippe Chavrier. "A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions." Journal of Cell Science 115, no. 14 (July 15, 2002): 2867–79. http://dx.doi.org/10.1242/jcs.115.14.2867.
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We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.
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Kostenko, Elena V., Oyenike O. Olabisi, Sutapa Sahay, Pedro L. Rodriguez, and Ian P. Whitehead. "Ccpg1, a Novel Scaffold Protein That Regulates the Activity of the Rho Guanine Nucleotide Exchange Factor Dbs." Molecular and Cellular Biology 26, no. 23 (September 25, 2006): 8964–75. http://dx.doi.org/10.1128/mcb.00670-06.
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ABSTRACT Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.
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Kubiseski, Terrance J., Joe Culotti, and Tony Pawson. "Functional Analysis of the Caenorhabditis elegans UNC-73B PH Domain Demonstrates a Role in Activation of the Rac GTPase In Vitro and Axon Guidance In Vivo." Molecular and Cellular Biology 23, no. 19 (October 1, 2003): 6823–35. http://dx.doi.org/10.1128/mcb.23.19.6823-6835.2003.
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ABSTRACT The Caenorhabditis elegans UNC-73B protein regulates axon guidance through its ability to act as a guanine nucleotide exchange factor (GEF) for the CeRAC/MIG-2 GTPases. Like other GEFs for Rho family GTPases, UNC-73B has a Dbl homology (DH) catalytic domain, followed by a C-terminal pleckstrin homology (PH) domain. We have explored whether the PH domain cooperates with the adjacent DH domain to promote UNC-73B GEF activity and axonal pathfinding. We show that the UNC-73B PH domain binds preferentially to monophosphorylated phosphatidylinositides in vitro. Replacement of residues Lys1420 and Arg1422 with Glu residues within the PH domain impaired this phospholipid binding but did not affect the in vitro catalytic activity of the DH domain. In contrast, a mutant UNC-73B protein with a Trp1502-to-Ala substitution in the PH domain still interacted with phosphorylated phosphatidylinositides but had lost its GEF activity. UNC-73B minigenes containing these mutations were microinjected into C. elegans and transferred to unc-73(e936) mutant worms. Unlike the wild-type protein, neither PH domain mutant was able to rescue the unc-73 axon guidance defect. These results suggest that the UNC-73B PH domain plays distinct roles in targeting and promoting GEF activity towards the Rac GTPase, both of which are important for the directed movements of motorneurons in vivo.
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Leung, T., X. Q. Chen, E. Manser, and L. Lim. "The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton." Molecular and Cellular Biology 16, no. 10 (October 1996): 5313–27. http://dx.doi.org/10.1128/mcb.16.10.5313.
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The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.
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Cong, Feng, Bing Yuan, and Stephen P. Goff. "Characterization of a Novel Member of the DOK Family That Binds and Modulates Abl Signaling." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8314–25. http://dx.doi.org/10.1128/mcb.19.12.8314.
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ABSTRACT A novel member of the p62 dok family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62 dok bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62 dok , DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62 dok , suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.
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Wolf, Ingrid, Brendan J. Jenkins, Yan Liu, Martina Seiffert, Joseph M. Custodio, Paul Young, and Larry R. Rohrschneider. "Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 231–44. http://dx.doi.org/10.1128/mcb.22.1.231-244.2002.
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ABSTRACT Using the FDC-P1 cell line expressing the exogenous macrophage colony-stimulating factor (M-CSF) receptor, Fms, we have analyzed the role of a new mammalian DOS/Gab-related signaling protein, called Gab3, in macrophage cell development of the mouse. Gab3 contains an amino-terminal pleckstrin homology domain, multiple potential sites for tyrosine phosphorylation and SH2 domain binding, and two major polyproline motifs potentially interacting with SH3 domains. Among the growing family of Gab proteins, Gab3 exhibits a unique and overlapping pattern of expression in tissues of the mouse compared with Gab1 and Gab2. Gab3 is more restricted to the hematopoietic tissues such as spleen and thymus but is detectable at progressively lower levels within heart, kidney, uterus, and brain. Like Gab2, Gab3 is tyrosine phosphorylated after M-CSF receptor stimulation and associates transiently with the SH2 domain-containing proteins p85 and SHP2. Overexpression of exogenous Gab3 in FD-Fms cells dramatically accelerates macrophage differentiation upon M-CSF stimulation. Unlike Gab2, which shows a constant mRNA expression level after M-CSF stimulation, Gab3 expression is initially absent or low in abundance in FD cells expressing the wild-type Fms, but Gab3 mRNA levels are increased upon M-CSF stimulation. Moreover, M-CSF stimulation of FD-FmsY807F cells (which grow but do not differentiate) fails to increase Gab3 expression. These results suggest that Gab3 is important for macrophage differentiation and that differentiation requires the early phosphorylation of Gab2 followed by induction and subsequent phosphorylation of Gab3.
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Pyrpassopoulos, Serapion, Henry Shuman, and E. Michael Ostap. "Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes." Molecular Biology of the Cell 28, no. 23 (November 7, 2017): 3315–22. http://dx.doi.org/10.1091/mbc.e17-05-0324.
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KIF16B is a highly processive kinesin-3 family member that participates in the trafficking and tubulation of early endosomes along microtubules. KIF16B attaches to lipid cargoes via a PX motif at its C-terminus, which has nanomolar affinity for bilayers containing phosphatidylinositol-3-phosphate (PI[3]P). As the PX domain has been proposed to be a primary mechanical anchor for the KIF16B-cargo attachment, we measured the adhesion forces and detachment kinetics of the PX domain as it interacts with membranes containing 2% PI(3)P and 98% phosphatidylcholine. Using optical tweezers, we found that the adhesion strength of a single PX domain ranged between 19 and 54 pN at loading rates between 80 and 1500 pN/s. These forces are substantially larger than the interaction of the adhesion of a pleckstrin homology domain with phosphatidylinositol 4,5-bisphosphate. This increased adhesion is the result of the membrane insertion of hydrophobic residues adjacent to the PI(3)P binding site, in addition to electrostatic interactions with PI(3)P. Attachment lifetimes under load decrease monotonically with force, indicating slip-bond behavior. However, the lifetime of membrane attachment under load appears to be well matched to the duration of processive motility of the KIF16B motor, indicating the PX domain is a suitable mechanical anchor for intracellular transport.
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Cheng, Jing, Peter C. Lucas, and Linda M. McAllister-Lucas. "Canonical and Non-Canonical Roles of GRK2 in Lymphocytes." Cells 10, no. 2 (February 3, 2021): 307. http://dx.doi.org/10.3390/cells10020307.
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G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative signaling node in a variety of biological processes ranging from cell growth and proliferation to migration and chemotaxis. As such, GRK2 is now implicated as playing a role in the molecular pathogenesis of a broad group of diseases including heart failure, cancer, depression, neurodegenerative disease, and others. In addition to its long-known canonical role in the phosphorylation and desensitization of G protein-coupled receptors (GPCRs), recent studies have shown that GRK2 also modulates a diverse array of other molecular processes via newly identified GRK2 kinase substrates and via a growing number of protein-protein interaction binding partners. GRK2 belongs to the 7-member GRK family. It is a multidomain protein containing a specific N-terminal region (referred to as αN), followed by a regulator of G protein signaling homology (RH) domain, an AGC (Protein kinase A, G, C serine/threonine kinase family) kinase domain, and a C-terminal pleckstrin homology (PH) domain. GPCRs mediate the activity of many regulators of the immune system such as chemokines and leukotrienes, and thus GRK proteins may play key roles in modulating the lymphocyte response to these factors. As one of the predominant GRK family members expressed in immune cells, GRK2′s canonical and noncanonical actions play an especially significant role in normal immune cell function as well as in the development and progression of disorders of the immune system. This review summarizes our current state of knowledge of the roles of GRK2 in lymphocytes. We highlight the diverse functions of GRK2 and discuss how ongoing investigation of GRK2 in lymphocytes may inform the development of new therapies for diseases associated with lymphocyte dysregulation.
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JENSEN, Rikke B., Tanja LACOUR, Jakob ALBRETHSEN, Michael NIELSEN, and Karen SKRIVER. "FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains." Biochemical Journal 359, no. 1 (September 24, 2001): 165–73. http://dx.doi.org/10.1042/bj3590165.
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Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)1(R/K)HHCR6 (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His4 and Arg6 of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P2, the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn4,Tyr6 → His4,Arg6). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.
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Bi, Feng, Balazs Debreceni, Kejin Zhu, Barbara Salani, Alessandra Eva, and Yi Zheng. "Autoinhibition Mechanism of Proto-Dbl." Molecular and Cellular Biology 21, no. 5 (March 1, 2001): 1463–74. http://dx.doi.org/10.1128/mcb.21.5.1463-1474.2001.
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ABSTRACT The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.
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Cuthbert, Ellen J., Kathryn K. Davis, and James E. Casanova. "Substrate specificities and activities of AZAP family Arf GAPs in vivo." American Journal of Physiology-Cell Physiology 294, no. 1 (January 2008): C263—C270. http://dx.doi.org/10.1152/ajpcell.00292.2007.
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The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401–413, 2004), are characterized by the presence of at least one NH2-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599–610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH2-terminal sterile-α motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH2-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.
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Snider, Chloe E., Alaina H. Willet, HannahSofia T. Brown, Jun-Song Chen, Joshua M. Evers, and Kathleen L. Gould. "Fission yeast Opy1 is an endogenous PI(4,5)P2 sensor that binds to the phosphatidylinositol 4-phosphate 5-kinase Its3." Journal of Cell Science 133, no. 23 (November 10, 2020): jcs247973. http://dx.doi.org/10.1242/jcs.247973.
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ABSTRACTPhosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in Schizosaccharomyces pombe. While examining potential PI(4,5)P2-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for S. pombe Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P2 levels. Our data indicate that although Opy1 senses PM PI(4,5)P2 levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P2 levels, a striking difference from its Saccharomyces cerevisiae homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P2. Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.
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O'Callaghan, Dermott W., Lee P. Haynes, and Robert D. Burgoyne. "High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate." Biochemical Journal 391, no. 2 (October 10, 2005): 231–38. http://dx.doi.org/10.1042/bj20051001.
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Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (Kd 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1–14)–ECFP (enhanced cyan fluorescent protein) or NCS-1–ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase δ1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.
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Takahashi-Tezuka, Mariko, Yuichi Yoshida, Toshiyuki Fukada, Takuya Ohtani, Yojiro Yamanaka, Keigo Nishida, Koichi Nakajima, Masahiko Hibi, and Toshio Hirano. "Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 4109–17. http://dx.doi.org/10.1128/mcb.18.7.4109.
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ABSTRACT Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation.
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Chen, Yujie, Wen Zhang, Bin Chen, Ying Liu, Yuhui Dong, Aimin Xu, and Quan Hao. "Crystal structure of human APPL BAR-PH heterodimer reveals a flexible dimeric BAR curve: implication in mutual regulation of endosomal targeting." Biochemical Journal 477, no. 24 (December 24, 2020): 4769–83. http://dx.doi.org/10.1042/bcj20200438.
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The APPL (adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif) family consists of two isoforms, APPL1 and APPL2. By binding to curved plasma membrane, these adaptor proteins associate with multiple transmembrane receptors and recruit various downstream signaling components. They are involved in the regulation of signaling pathways evoked by a variety of extracellular stimuli, such as adiponectin, insulin, FSH (follicle stimulating hormone), EGF (epidermal growth factor). And they play important roles in cell proliferation, apoptosis, glucose uptake, insulin secretion and sensitivity. However, emerging evidence suggests that APPL1 and APPL2 perform different or even opposite functions and the underlying mechanism remains unclear. As APPL proteins can either homodimerize or heterodimerize in vivo, we hypothesized that heterodimerization of APPL proteins might account for the mechanism. By solving the crystal structure of APPL1–APPL2 BAR-PH heterodimer, we find that the overall structure is crescent-shaped with a longer curvature radius of 76 Å, compared with 55 Å of the APPL1 BAR-PH homodimer. However, there is no significant difference of the curvature between APPL BAR-PH heterodimer and APPL2 homodimer. The data suggest that the APPL1 BAR-PH homodimer, APPL2 BAR-PH homodimer and APPL1/APPL2 BAR-PH heterodimer may bind to endosomes of different sizes. Different positive charge distribution is observed on the concave surface of APPL BAR-PH heterodimer than the homodimers, which may change the affinity of membrane association and subcellular localization. Collectively, APPL2 may regulate APPL1 function through altering the preference of endosome binding by heterodimerization.
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Kinoshita, Riko, Yuta Homma, and Mitsunori Fukuda. "Rab35–GEFs, DENND1A and folliculin differentially regulate podocalyxin trafficking in two- and three-dimensional epithelial cell cultures." Journal of Biological Chemistry 295, no. 11 (January 28, 2020): 3652–63. http://dx.doi.org/10.1074/jbc.ra119.011646.
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Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin–Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain–containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
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Chen, Wei, Nan Li, Taoyong Chen, Yanmei Han, Changfei Li, Yuzhen Wang, Weigang He, Lihuang Zhang, Tao Wan, and Xuetao Cao. "The Lysosome-associated Apoptosis-inducing Protein Containing the Pleckstrin Homology (PH) and FYVE Domains (LAPF), Representative of a Novel Family of PH and FYVE Domain-containing Proteins, Induces Caspase-independent Apoptosis via the Lysosomal-Mitochondrial Pathway." Journal of Biological Chemistry 280, no. 49 (September 27, 2005): 40985–95. http://dx.doi.org/10.1074/jbc.m502190200.
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Gueller, Saskia, Sigal Gery, and H. Phillip Koeffler. "Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein." Blood 110, no. 11 (November 16, 2007): 2213. http://dx.doi.org/10.1182/blood.v110.11.2213.2213.
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Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.
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Chen, Wei, Nan Li, Taoyong Chen, Yanmei Han, Changfei Li, Yuzhen Wang, Weigang He, Lihuang Zhang, Tao Wan, and Xuetao Cao. "Withdrawal: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway." Journal of Biological Chemistry 296 (January 2021): 100764. http://dx.doi.org/10.1016/j.jbc.2021.100764.
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Zulkiflee, Nurul Syeefa, Siti Amilia Awang, Woo Xian Ming, Muhammad Fauzan Wira’i Kamilan, M. Yuveneshwari Mariappan, and Tan Jen Kit. "In Silico Docking of Vitamin E Isomers on Transport Proteins." Current Computer-Aided Drug Design 16, no. 4 (September 3, 2020): 467–72. http://dx.doi.org/10.2174/1573409915666190614113733.
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Background: Vitamin E is comprised of α, β, γ and δ-tocopherols (Ts) and α, β, γ and δ- tocotrienols (T3s). Vitamin E has neuroprotective antioxidant, anti-cancer, and cholesterol-lowering effects. Intracellular trafficking of these isomers remains largely unknown, except for αT which is selectively transported by αT transfer protein (αTTP). Objective: This study aimed to determine the binding of vitamin E isomers on transport proteins using in silico docking. Methods: Transport proteins were selected using AmiGo Gene Ontology tool based on the same molecular function annotation as αTTP. Protein structures were obtained from the Protein Data Bank. Ligands structures were obtained from ZINC database. In silico docking was performed using SwissDock. Results and Discussion: A total of 6 transport proteins were found: SEC14-like protein 2, glycolipid transfer protein (GLTP), pleckstrin homology domain-containing family A member 8, collagen type IV alpha-3-binding protein, ceramide-1-phosphate transfer protein and afamin. Compared with other transport proteins, αTTP had the highest affinities for all isomers except βT3. Binding order of vitamin E isomers toward αTTP was γT > βT > αT > δT > αT3 > γT3 > δT3 > βT3. GLTP had a higher affinity for tocotrienols than tocopherols. βT3 bound stronger to GLTP than αTTP. Conclusion: αTTP remained as the most preferred transport protein for most of the isomers. The binding affinity of αT toward αTTP was not the highest than other isomers suggested that other intracellular trafficking mechanisms of these isomers may exist. GLTP may mediate the intracellular transport of tocotrienols, especially βT3. Improving the bioavailability of these isomers may enhance their beneficial effects to human.
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Na, Woojin, Eun-Jung Lee, Min-Kyung Kang, Yun-Ho Kim, Dong Yeon Kim, Hyeongjoo Oh, Soo-Il Kim, Su Yeon Oh, and Young-Hee Kang. "Aesculetin Inhibits Osteoclastic Bone Resorption through Blocking Ruffled Border Formation and Lysosomal Trafficking." International Journal of Molecular Sciences 21, no. 22 (November 13, 2020): 8581. http://dx.doi.org/10.3390/ijms21228581.
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For the optimal resorption of mineralized bone matrix, osteoclasts require the generation of the ruffled border and acidic resorption lacuna through lysosomal trafficking and exocytosis. Coumarin-type aesculetin is a naturally occurring compound with anti-inflammatory and antibacterial effects. However, the direct effects of aesculetin on osteoclastogenesis remain to be elucidated. This study found that aesculetin inhibited osteoclast activation and bone resorption through blocking formation and exocytosis of lysosomes. Raw 264.7 cells were differentiated in the presence of 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and treated with 1–10 μM aesculetin. Differentiation, bone resorption, and lysosome biogenesis of osteoclasts were determined by tartrate-resistance acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, immunocytochemical analysis, and LysoTracker staining. Aesculetin inhibited RANKL-induced formation of multinucleated osteoclasts with a reduction of TRAP activity. Micromolar aesculetin deterred the actin ring formation through inhibition of induction of αvβ3 integrin and Cdc42 but not cluster of differentiation 44 (CD44) in RANKL-exposed osteoclasts. Administering aesculetin to RANKL-exposed osteoclasts attenuated the induction of autophagy-related proteins, microtubule-associated protein light chain 3, and small GTPase Rab7, hampering the lysosomal trafficking onto ruffled border crucial for bone resorption. In addition, aesculetin curtailed cellular induction of Pleckstrin homology domain-containing protein family member 1 and lissencephaly-1 involved in lysosome positioning to microtubules involved in the lysosomal transport within mature osteoclasts. These results demonstrate that aesculetin retarded osteoclast differentiation and impaired lysosomal trafficking and exocytosis for the formation of the putative ruffled border. Therefore, aesculetin may be a potential osteoprotective agent targeting RANKL-induced osteoclastic born resorption for medicinal use.
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Gueller, Saskia, Helen S. Goodridge, Hongtao Xing, Sigal Gery, Hubert Serve, and H. Phillip Koeffler. "Adaptor Protein Lnk Inhibits C-Fms Mediated Macrophage Function." Blood 112, no. 11 (November 16, 2008): 3555. http://dx.doi.org/10.1182/blood.v112.11.3555.3555.
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Abstract The macrophage colony-stimulating factor receptor (c-Fms) plays an important role in proliferation, differentiation and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. One such protein is the adaptor Lnk that negatively regulates several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain and a pleckstrin homology domain. The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. In this study, we investigated the ability of Lnk to interact and modulate the function of c-Fms. In order to determine if Lnk can bind to c-Fms, immunoprecipitation was performed with lysates from 293T cells co-transfected with the cDNAs for c-Fms and Lnk. Only after exposure to M-CSF, Lnk bound to c-Fms, and binding was dependent on an intact SH2 domain. To elucidate further if Lnk exhibits biological and functional effects on macrophages, we examined both in-vitro differentiated macrophages derived from the bone marrow and also macrophages harvested from peritoneum from Lnk deleted (KO) and wild type (WT) mice. These cells appeared to be at a similar stage of differentiation because expression levels of myeloid and macrophage surface markers such as F4/80, CD11b and CD11c were the same in both bone marrow-derived and peritoneum-derived macrophages from Lnk KO and WT mice. Clonogenic assays demonstrated that the number of M-CFUs in the bone marrow were elevated in Lnk KO as compared to WT mice. Furthermore, the M-CSF-induced phosphorylation of AKT in these Lnk KO macrophages was increased and prolonged compared to WT macrophages. This was associated with prominent up-regulation of c-Fms in macrophages from Lnk KO mice. We found that Lnk additionally had several functional effects on bone marrow-derived macrophages. Production of reactive oxygen species (ROS) was dramatically increased in a M-CSF-dependent manner in Lnk KO macrophages upon stimulation with zymosan. In addition, knock-out of Lnk led to altered cytokine production of macrophages: Stimulation with zymosan caused increased levels of TNFalpha and IL-6 in the KO cells, while bacterial lipoproteins (Pam3CSK4) decreased levels of TNFalpha in KO compared to WT macrophages. Last, Lnk inhibited M-CSF-induced migration of macrophages in the Boyden chamber as Lnk KO macrophages showed a significantly higher migration capacity than WT macrophages. In summary, we show for the first time that Lnk can bind to c-Fms and can blunt the stimulation of M-CSF. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.
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Shereck, Evan, Nancy Day, Catherine McGuinn, Prakash Satwani, Carmella van de Ven, Janet Ayello, David Crockett, et al. "Immunophenotypic and Proteomic Characterization of Cord Blood (CB) CD56dim Versus Peripheral Blood (PB) CD56dim NK Cells." Blood 110, no. 11 (November 16, 2007): 3879. http://dx.doi.org/10.1182/blood.v110.11.3879.3879.
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Abstract Background: Neonates may succumb to serious infection due to deficiencies in adaptive cellular immunity, however, little is known regarding the immaturity of CB NK cell innate immunity (Satwani/Cairo et al Biol Neonate 2005). NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56brigh t (10%, mediator) (Shankaran et al Nature 2001). NK subsets’ function is based on their repertoire of NK receptors (NKRs) (Moretta et al Annu Rev Immunol 2001). Furthermore, the amount of CB NK cells is significantly low in cryopreserved CB for CBT (Cairo et al Transfusion 2005), however, they may play an important role in the graft vs leukemia (GVL) effect post CBT. We recently demonstrated the ability to expand CB into NK phenotype ex-vivo with profound NK cytotoxic activity (Ayello/Cairo et al BBMT 2006). Objectives: To characterize and compare CB vs. PB NK CD56dim NKR and protein expression. Methods: We positively selected CB vs. PB CD56+ cells using immunomagnetic beads (Miltenyi) and sorted into CD3−/CD56bright and CD3−/CD56dim subsets. NKR expression was evaluated using CD16, CD158a {KIR2DL1}, CD158a,h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46. Additionally, quantitative proteomic analysis was performed using extracts of CB and PB CD56Dim cells using cleavable ICAT labeling followed by multi-dimensional liquid chromatography and tandem mass spectrometry (MS/MS) (Lim et al Lab Invest 2004). Results: CB vs. PB CD56dim showed significant increased expression of NKG2A (81.34 ± 1.51 vs. 54.09 ± 4.56, p < 0.03) and NKG2D (94.08 ± 2.36 vs. 77.43 ± 3.93, p < 0.035). There was no significant difference in NKR expression of CD16, KIR2DL1, KIR2DS1, KIR2DL2, CD161, NKG2C, Nkp44, and NKp46 seen in CB vs. PB CD56dim. CB CD56dim vs. CD56bright had significant increased expression of FcgrIII (91.17 ± 1.95 vs. 28.73 ± 11.23, p < 0.03) and KIR2DL2 (31.88 ± 4.39 vs. 3.08 ± 0.99, p < 0.02). Furthermore, there were 33 and 37 proteins over and under expressed by ≥ 2 fold between CB vs. PB CD56dim NK cells, respectively. CB CD56dim overexpressed functional proteins were 46% binding, 17% catalytic, 15% signaling, 15% transcription, 3% enzyme, 2% structural, and 2% transport. CB CD56dim underexpressed Inositol 1,4,5-triphosphate receptor type 3, mitogen-activated protein kinase 5, and natural cytotoxicity triggering receptor 3 precursor by 8.33, 6.67, and 2.5 fold decrease, respectively. CB CD56dim overexpressed neurogenic locus notch homolog protein 2 precursor, pleckstrin homology domain-containing family A member 1, and transcriptional repressor NF-X1 by 16.67, 7.14, and 5.26 fold increase, respectively. Conclusion: While there are many similiarities in CB vs. PB CD56dim, CB CD56dim appear to be quite mature in development compared to other CB immune cell subsets and in fact have overexpression of a significant number of proteins including NKG2D, NKG2A, and several (33) important functional proteins. These studies suggest that CB CD56dim NK cells are similar in development to PB CD56dim NK cells and may contribute to the GVL effect post UCBT.
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"Pleckstrin homology domain containing family O member 1 ( PLEKHO1 ; CKIP-1 )." Science-Business eXchange 5, no. 6 (February 2012): 157. http://dx.doi.org/10.1038/scibx.2012.157.
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"Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway." Journal of Biological Chemistry 295, no. 26 (June 26, 2020): 8877. http://dx.doi.org/10.1074/jbc.ec120.014564.
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