YXXL Motif Research Articles

YXXL Motifs in SH2-Bॆ Are Phosphorylated by JAK2, JAK1, and Platelet-derived Growth Factor Receptor and Are Required for Membrane Ruffling

SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.

The Conserved Process of TCR/CD3 Complex Down-Modulation by SIV Nef Is Mediated by the Central Core, Not Endocytic Motifs

The Nef protein of Simian immunodeficiency virus (SIV) associates with multiple T lymphocyte signaling proteins, including the T cell receptor (TCR) ζ chain. We demonstrate here that these interactions are conserved and highly specific. Nefs derived from genetically diverse strains of SIV (SIV mac239, SIV smmPBj, and SIV smmΔB670) all interacted with TCR ζ on two separate domains, referred to as SIV Nef interaction domains (SNIDs), as examined in both yeast two-hybrid and glutathione- S-transferase (GST) fusion protein pull-down assays. Multiple HIV-1 Nefs were examined and none interacted with TCR ζ. In contrast, HIV-2 UC1 Nef, similar to SIV Nef, interacted with TCR ζ on two domains, although only the SIV Nefs potently reduced cell-surface expression of the TCR/CD3 complex in T cells. In addition, we examined the abilities of SIV, HIV-2, and HIV-1 Nefs to interact with the cytoplasmic domains of other signaling molecules including CD3ϵ, CD3γ, and FcϵRIγ, which also contain YxxL motifs, and determined that SIV and HIV-2 Nefs interacted only with TCR ζ, whereas HIV-1 Nef did not interact with any signal-transducing cytoplasmic domain examined. Last, to gain further insight into the mechanism by which Nef down-modulates the TCR/CD3 complex, we mutated or deleted regions on Nef involved in endocytosis, localization of Nef to the plasma membrane, interaction with cellular kinases, or that were conserved among multiple strains of SIV. Mutation of the myristoylation site and a conserved region surrounding a putative PKC phosphorylation site were the only mutations that abrogated Nef-mediated down-modulation of the TCR/CD3 complex. These findings demonstrate there is a spectrum of associations between SIV, HIV-2, and HIV-1 Nefs, and the TCR/CD3 complex, and suggest that down-modulation of the TCR/CD3 complex occurs via association with subsets of cellular proteins that are different from those involved in CD4 and CD28 down-modulation.

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.

The YXXL Motif, but Not the Two NPXY Motifs, Serves as the Dominant Endocytosis Signal for Low Density Lipoprotein Receptor-related Protein

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission.

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.

Volume Expansion Stimulates p72syk and p56lyn in Skate Erythrocytes

Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with 32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety.

Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly.

We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the alpha adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.

Low-voltage separation of phosphoamino acids by silica gel thin-layer electrophoresis in a DNA electrophoresis cell.

BioTechniquesVol. 24, No. 3 BenchmarksOpen AccessLow-Voltage Separation of Phosphoamino Acids by Silica Gel Thin-Layer Electrophoresis in a DNA Electrophoresis CellShane C. Hardin & Stephen M. WolniakShane C. HardinUniversity of Maryland, College Park, MD, USASearch for more papers by this author & Stephen M. Wolniak*Address correspondence to Stephen M. Wolniak, Department of Microbiology, University of Maryland, H.J. Patterson Hall, College Park, MD 20742, USA. Internet: E-mail Address: sw36@umail.umd.eduUniversity of Maryland, College Park, MD, USASearch for more papers by this authorPublished Online:15 Aug 2018https://doi.org/10.2144/98243bm01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByNeutral Sphingomyelinase 2 (nSMase2) Is a Phosphoprotein Regulated by Calcineurin (PP2B)Journal of Biological Chemistry, Vol. 285, No. 14Post‐translational modification of an R2R3‐MYB transcription factor by a MAP Kinase during xylem development10 August 2009 | New Phytologist, Vol. 183, No. 4Taking a Walk on the Wild Side with Planar Electrochromatography and Thin‐Layer Electrophoresis: Of Peptides, Proteins, and Proteomics6 February 2007 | Journal of Liquid Chromatography & Related Technologies, Vol. 29, No. 7-8Dileucine and YXXL Motifs in the Cytoplasmic Tail of the Bovine Leukemia Virus Transmembrane Envelope Protein Affect Protein Expression on the Cell SurfaceJournal of Virology, Vol. 78, No. 15Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-123 June 2003 | European Journal of Biochemistry, Vol. 270, No. 14MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1Journal of Biological Chemistry, Vol. 277, No. 43The protein kinases AtMAP3Kε1 and BnMAP3Kε1 are functional homologues of S. pombe cdc7p and may be involved in cell division23 December 2001 | The Plant Journal, Vol. 26, No. 6Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 712015 December 2003 | European Journal of Biochemistry, Vol. 268, No. 6 Vol. 24, No. 3 Follow us on social media for the latest updates Metrics Downloaded 142 times History Published online 15 August 2018 Published in print March 1998 Information© 2018 Author(s)PDF download

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.

Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.

Varicella-zoster virus (VZV) encodes a cell surface Fc receptor, glycoprotein gE. VZV gE has previously been shown to display several features common to nonviral cell surface receptors. Most recently, VZV gE was reported to be tyrosine phosphorylated on a dimeric form (J. K. Olson, G. A. Bishop, and C. Grose, J. Virol. 71:110-119, 1997). Thereafter, attention focused on the ability of VZV gE to undergo receptor-mediated endocytosis. The current transient transfection studies demonstrated by confocal microscopy and internalization assays that VZV gE was endocytosed when expressed in HeLa cells. Endocytosis of gE was shown to be dependent on clathrin-coated vesicle formation within the cells. Subsequent colocalization studies showed that endocytosis of VZV gE closely mimicked endocytosis of the transferrin receptor. The gE cytoplasmic tail and more specifically tyrosine residue 582 were determined by mutagenesis studies to be important for efficient internalization of the protein; this tyrosine residue is part of a conserved YXXL motif. The amount of gE internalized at any given time reached a steady state of 32%. In addition, like the transferrin receptor, internalized gE recycled to the cell surface. The finding of gE endocytosis provided insight into earlier documentation of gE serine/threonine and tyrosine phosphorylation, since these phosphorylation events may serve as sorting signals for internalized receptors. Taken together with the previous discovery that both human and simian immunodeficiency virus envelope proteins can undergo endocytosis, the gE findings suggest that endocytosis of envelope components may be a posttranslational regulatory mechanism among divergent families of enveloped viruses.

Identification of a nef allele that causes lymphocyte activation and acute disease in Macaque monkeys

Residues 17 and 18 In nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRP-SGDLYERLLRARGETYGRLLGEVEDGYSOSP from residues 10–43. The YXXL motifs In this context match very well with consensus sequences for SH2 binding domains and are similar to ones present In nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting peripheral blood mononuclear cell cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pigtailed monkeys characterized by severe diarrhea, rash, and extensive lymphoid proliferation In the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with arc In cotransfected COS cells, and both 60 kDa arc and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype, and they provide evidence of a role for nef in signal transduction and cellular activation.

The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads.

The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation.

The Drosophila Insulin Receptor Contains a Novel Carboxyl-terminal Extension Likely to Play an Important Role in Signal Transduction

The nucleic acid and deduced amino acid sequence of the Drosophila insulin receptor homologue (dir) has been determined. The coding sequence of dir is contained within 10 exons spanning less than 8 kilobase pairs of genomic DNA. The deduced amino acid sequence of the dir encodes a protein of 2148 amino acids, larger than the human insulin receptor due to amino- and carboxyl-terminal extensions. The overall level of amino acid identity between the DIR and human insulin and insulin-like growth factor-I receptors is 32.5 and 33.3%, respectively. Higher levels of identity are found in exon 2 (45 and 43%, respectively) and in the beta subunit (50 and 48%, respectively), and the positions of most cysteine residues in the alpha subunit cysteine-rich domain are conserved. A novel, 400-amino acid, carboxyl-terminal extension contains 9 tyrosine residues, four of which are present in YXXM or YXXL motifs, suggesting that they function as binding sites for SH2 domain-containing signaling proteins. The presence of multiple putative SH2 domain binding sites in the DIR represents a significant difference from its mammalian homologues and suggests that, unlike the human insulin and insulin-like growth factor-I receptors, the DIR forms stable complexes with signaling molecules as part of its signal transduction mechanism.

The cytoplasmic tail of the T cell receptor ζ chain is required for signaling via CD26

The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymphocytes and is involved in several aspects of T cell function. Activation via CD26 requires the expression of the T cell receptor (TcR)/CD3 complex. Here we have investigated the role of the TcR zeta chain for T cell activation via CD26. T cell hybridomas expressing TcR with various deletions in the CD3 zeta chain were transfected with a CD26 cDNA and the response of the transfected cells to anti-CD26 monoclonal antibodies was tested. Our data show that the zeta chain is essential and that at least one YXXL motif in the cytoplasmic tail of the zeta chain is required for CD26-mediated signaling. Other TcR components do not replace the zeta chain.