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Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation  [PDF]
Kyu-Hee Lee,Won-Kyung Ho,Suk-Ho Lee
Frontiers in Cellular Neuroscience , 2013, DOI: 10.3389/fncel.2013.00014
Abstract: We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.
Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity  [PDF]
Sandesh Subramanya,Kojo Mensa-Wilmot
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008538
Abstract: Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.
Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity
Sushma J Bhakta, Liang Shang, Jessica L Prince, Daniel T Claiborne, Eric Hunter
Retrovirology , 2011, DOI: 10.1186/1742-4690-8-37
Abstract: All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.The envelope glycoprotein (Env) cytoplasmic domain (CD) is a key determinant in the replication of Human Immunodeficiency Virus type I (HIV-1) at two pivotal steps: (i) at the point of viral assembly, where Env must be incorporated into budding virions, and (ii) at the stage of viral entry into host target cells. The Env CD has been shown through both genetic and biochemical approaches to interact with domains of Gag during assembly [1-3], interact with cellular components during intracellular transport [4-7], modulate the fusogenicity of the Env complex both in the cell and within the virion [4,8,9], and regulate the cell surface expression of Env [10-13]. However, exactly which Env CD sequences mediate these phenotypically important roles remains t
Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs
Hans-Jürg Monstein, Anneli Karlsson, Anna Ryberg, Kurt Borch
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-35
Abstract: MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy.Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.Helicobacter pylori is a microaerophilic Gram-negative bacterium that chronically infects the gastric mucosa. It is recognised as a human pathogen associated not only with chronic gastritis [1], but also with peptic ulcer [2] and gastric cancer [3]. A commonly used molecular marker of H. pylori virulence is the cagA gene (cytotoxin-associated gene) [4], which is a part of the 40 kb Cag-Pathogenicity Island (cag-PAI) [5]. The CagA cytotoxin is directly injected into epithelial cells by a type IV secretion system, encoded by genes located in the cag-PAI [6-8]. In the host cell, CagA localises to the plasma membrane and undergoes phosphorylation on specific tyrosine residues within repeating penta amino acid Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, present at the C-terminus of the protein [9,10]. The C-terminal part, which contains the EPIYA motifs, has been shown to be highly variable, as opposed to the highly conserved N-terminal part [7,11-13]. CagA EPIYA motifs are defined as EPIYA-A, -B, -C, and -D, according to the amino acid sequences that surround the EPIYA sequence [10,13,14]. CagA
Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome
Tiwari, Santosh K.;Sharma, Vishwas;Sharma, Varun Kumar;Gopi, Manoj;Saikant, R;Nandan, Amrita;Bardia, Avinash;Gunisetty, Sivaram;Katikala, Prasanth;Habeeb, Md. Aejaz;Khan, Aleem A.;Habibullah, C.M.;
Genetics and Molecular Biology , 2011, DOI: 10.1590/S1415-47572011005000003
Abstract: the population of india harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. several phylogenetic studies involving mitochondrial dna and y chromosomal variation have demonstrated europeans to have been the first settlers in india. nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the austro-asiatic group, prior to the europeans. thus, the aim was to investigate pre-historic colonization of india by anatomically modern humans, using conserved stretches of five amino acid (epiya) sequences in the caga gene of helicobacter pylori. simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (tpms), in 32 h. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. high resolution sequence analysis of the above described genes was performed. the nucleotide sequences obtained were translated into amino acids using mega (version 4.0) software for epiya. an mj-network was constructed for obtaining tpm haplotypes by using network (version 4.5) software. the findings of the study suggest that indian h. pylori strains share a common ancestry with europeans. no specific association of haplotypes with the outcome of disease was revealed through additional network analysis of tpms.
EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination  [PDF]
Dipanjan Dutta,Sayan Chakraborty,Chirosree Bandyopadhyay,Mohanan Valiya Veettil,Mairaj Ahmed Ansari,Vivek Vikram Singh,Bala Chandran
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003510
Abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.
The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking
Vincent Blot, Sandra Lopez-Vergès, Marie Breton, Claudine Pique, Clarisse Berlioz-Torrent, Marie-Pierre Grange
Retrovirology , 2006, DOI: 10.1186/1742-4690-3-62
Abstract: We used a CD25 (Tac) chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs) of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN) toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors.This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly.Retroviruses are surrounded by a lipid envelope acquired by the virus from cellular membranes through a budding process. Anchored in this lipid envelope are the viral envelope glycoproteins (Env), which are heterodimers between a transmembrane subunit (TM) and a covalently or non-covalently attached extracellular subunit (named SU for surface). Both subunits emerge from the cleavage of a single type-1 transmembrane envelope glycoprotein precursor (for review on retrovirus structural protein synthesis, see [1].The Gag proteins precursor, simply referred to here as Gag, is the only viral structural protein that is both necessary and sufficient to produce virus-like particles (VLPs) by budding into the extracellular medium, even in the absence of Env [2,3]. However, VLPs devoid of Env are non infectious since Env glycoproteins are necessary for the attachment of the virions to their receptor(s) and subsequent fusion of
Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with μ2  [PDF]
Jun Sung Lee, Il Shin Kim, Jung Hwan Kim, Wonhwa Cho, Pann-Ghill Suh, Sung Ho Ryu
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007090
Abstract: Background Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors. Methodology/Principal Findings Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. Conclusions/Significance These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.
Motifs and cis-regulatory modules mediating the expression of genes co-expressed in presynaptic neurons
Rui Liu, Sridhar Hannenhalli, Maja Bucan
Genome Biology , 2009, DOI: 10.1186/gb-2009-10-7-r72
Abstract: Using unbiased in vivo and in vitro screens, we characterized the cis elements regulating the Rab3A gene, which is expressed abundantly in presynaptic neurons. A set of identified regulatory elements of the Rab3A gene corresponded to the defined Rab3A multi-species conserved elements. In order to identify clusters of enriched transcription factor binding sites, for example, cis-regulatory modules, we analyzed intergenic multi-species conserved elements in the vicinity of nine presynaptic genes, including Rab3A, that are highly and specifically expressed in brain regions. Sixteen transcription factor binding motifs were over-represented in these multi-species conserved elements. Based on a combined occurrence for these enriched motifs, multi-species conserved elements in the vicinity of 107 previously identified presynaptic genes were scored and ranked. We then experimentally validated the scoring strategy by showing that 12 of 16 (75%) high-scoring multi-species conserved elements functioned as neuronal enhancers in a cell-based assay.This work introduces an integrative strategy of comparative genomics, experimental, and computational approaches to reveal aspects of a regulatory network controlling neuronal-specific expression of genes in presynaptic neurons.Synaptic transmission, the crucial process that enables information transfer in the nervous system, is a series of events in which neurotransmitters are released via exocytosis from presynaptic neurons and taken up by postsynaptic neurons. In presynaptic neurons, synaptic vesicles facilitate uptake of neurotransmitters and dock at the active zone of the plasma membrane. In response to calcium signaling, vesicles rapidly fuse with the plasma membrane and release neurotransmitters by exocytosis. The vesicles are recycled by subsequent endocytosis. These events are orchestrated by multiple protein complexes [1,2]. For example, one class of proteins is attached to the synaptic vesicle membrane, and is involved in ca
Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans  [PDF]
Didi Chen equal contributor,Youli Jian equal contributor,Xuezhao Liu,Yuanya Zhang,Jingjing Liang,Xiaying Qi,Hongwei Du,Wei Zou,Lianwan Chen,Yongping Chai,Guangshuo Ou,Long Miao,Yingchun Wang,Chonglin Yang
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003517
Abstract: Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.
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