Mutation Of Tyrosine Residue Research Articles

“Glyco-sulfo barcodes” regulate chemokine receptor function

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

The combination of ruxolitinib and Bcl-2/Mcl-1 inhibitors has a synergistic effect on leukemic cells carrying a SPAG9::JAK2 fusion.

JAK2 rearrangements can occur in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). Here, we performed functional analysis of the SPAG9::JAK2 fusion, which was identified in a pediatric patient with Ph-like ALL, to establish molecular targeted therapy. Ba/F3 cells expressing SPAG9::JAK2 generated by retroviral transduction (Ba/F3-SPAG9-JAK2), proliferated in the absence of IL-3, and exhibited constitutive phosphorylation of the tyrosine residues in the JAK2 kinase domain of the fusion protein and STAT3/STAT5. Mutation of tyrosine residues in the JAK2 kinase domain (SPAG9::JAK2 mut) abolished IL-3 independence, but had no influence on STAT3/STAT5 phosphorylation levels. Gene expression analysis revealed that Stat1 was significantly upregulated in Ba/F3-SPAG9-JAK2 cells. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells, suggesting that STAT1 is key for SPAG9::JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9::JAK2, but not SPAG9::JAK2 mut. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro, but was insufficient in vivo. Venetoclax (a BCL-2 inhibitor) or AZD5991 (an MCL-1 inhibitor) enhanced the effects of ruxolitinib on Ba/F3-SPAG9-JAK2 in vitro. These findings suggest that activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to SPAG9::JAK2-related aberrant growth promotion. BCL-2 or MCL-1 inhibition is a potential therapeutic option for B-ALL with SPAG9::JAK2 fusion.

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.

The Nitrated Proteome in Heart Mitochondria of the db/db Mouse Model: Characterization of Nitrated Tyrosine Residues in SCOT

A proteomic strategy combining 2DE, Western blot, and mass spectrometry was implemented to survey the status of tyrosine nitration in mouse heart mitochondria. Compared to normal mice, nitrated proteins in the heart mitochondria of the db/db mouse model were significantly augmented due to diabetic development. A total of 18 proteins were identified as the nitration targets. Of the nitrated proteins, succinyl-CoA:3-oxoacid CoA-transferase (SCOT) is a key enzyme involved in ketolysis and has yet to be explored how its catalysis is affected by nitration. We therefore initiated a systematic investigation toward the nitrated site(s) and the corresponding changes of SCOT catalysis. To monitor modification kinetics and nitrated residue(s), recombinant SCOT was incubated with peroxynitrite followed by examination of nitration development as well as catalytic activity changes. The nitration of recombinant SCOT steadily increased in response to increasing concentrations of peroxynitrite, while its catalysis was gradually attenuated. The nitrated sites of modified SCOT were further identified by LC-ESI-MS/MS. The MS/MS spectra indicated a +45 mass unit ion shift from [M + H](+) m/z at Tyr(4) and Tyr(76). Through site-directed mutagenesis, we found that mutation of tyrosine residues at Tyr(4) or Tyr(76) did not only significantly protect SCOT from peroxynitrite modification, but it also dramatically prevented loss of enzymatic activity. Taken together, these results indicate that the two tyrosine residues of SCOT are the priority sites attacked by NO, and their nitration status is a causal factor leading to inhibition of SCOT catalysis.

Src kinase‐dependent gating properties of single Ca2+ channels are altered by tyrosine nitration in colitis

Reduced L‐type Ca2+ ‐channel (LTCC) function contributes to colonic dysmotility during inflammation. We have previously shown that nitration of tyrosine residues within the carboxy terminus of LTCC (Y1837 and Y2134) impairs c‐Src kinase regulation during colitis. We examined the gating properties of single LTCC, using cell‐attached patch clamp mode, in mouse colon smooth muscle cells (control vs colitis‐TNBS) and CHO cells transfected with hCaV1.2b (wild type) or mutated hCaV1.2b (Y1837F:Y2134F). Inflammation significantly reduced LTCC ensemble average current (−0.33 vs −0.22 pA, P=0.05), without affecting the single‐channel properties: mean amplitude (−2.4pA), slope conductance (23 vs 27pS) or the open‐time constants (τ1: 1.6 vs 1.2; τ2: 6.1 vs 3.9 ms). However, the availability (slow gating) of LTCC was significantly reduced by inflammation (0.19 vs 0.06; P=0.02) without affecting the open probability of modal gating. Similar effects were seen with inhibition of c‐Src kinase by PP2 or by tyrosine nitration induced by SIN‐1+ONOO− combination in smooth muscle cells. Mutation of tyrosine residues (Y1837/2134F) in hCaV1.2b, which prevents c‐Src kinase regulation, resulted in diminished channel availability. This study demonstrates that src kinase‐ phosphorylation enhances the availability of LTCC, that is impaired by tyrosine nitration during colonic inflammation. Supported by NIH DK046367.

Organotypic specificity of key RET adaptor-docking sites in the pathogenesis of neurocristopathies and renal malformations in mice

The receptor tyrosine kinase ret protooncogene (RET) is implicated in the pathogenesis of several diseases and in several developmental defects, particularly those in neural crest-derived structures and the genitourinary system. In order to further elucidate RET-mediated mechanisms that contribute to these diseases and decipher the basis for specificity in the pleiotropic effects of RET, we characterized development of the enteric and autonomic nervous systems in mice expressing RET9 or RET51 isoforms harboring mutations in tyrosine residues that act as docking sites for the adaptors Plcgamma, Src, Shc, and Grb2. Using this approach, we found that development of the genitourinary system and the enteric and autonomic nervous systems is dependent on distinct RET-stimulated signaling pathways. Thus, mutation of RET51 at Y1062, a docking site for multiple adaptor proteins including Shc, caused distal colon aganglionosis reminiscent of Hirschsprung disease (HSCR). On the other hand, this mutation in RET9, which encodes an isoform that lacks the Grb2 docking site present in RET51, produced severe abnormalities in multiple organs. Mutations that abrogate RET-Plcgamma binding, previously shown to produce features reminiscent of congenital anomalies of kidneys or urinary tract (CAKUT) syndrome, produced only minor abnormalities in the nervous system. Abrogating RET51-Src binding produced no major defects in these systems. These studies provide insight into the basis of organotypic specificity and redundancy in RET signaling within these unique systems and in diseases such as HSCR and CAKUT.

Abrogation of High-Affinity IgE Receptor-Mediated Mast Cell Activation at the Effector Phase Prevents Contact Hypersensitivity to Oxazolone

Inflammatory mediators released from mast cells (MCs) through engagement of the high-affinity receptor for IgE (FcepsilonRI) have pivotal roles in chemical allergen-induced contact hypersensitivity (CHS) reactions, which suggests that the blockade of MC activation through FcepsilonRI stimulation may attenuate allergic contact dermatitis (CD). To address this possibility, we employed the following two approaches: (i) modulation of FcepsilonRI-mediated MC activation by introducing mutations in tyrosine residues of the FcepsilonRI beta-chain immunoreceptor tyrosine-based activation motif (ITAM) and (ii) blockade of FcepsilonRI-mediated MC activation employing a recombinant soluble ecto-domain of the human FcepsilonRIalpha-chain (rsFcepsilonRIalpha). In this study, we show that optimal MC activation through the FcepsilonRI beta-chain ITAM has essential roles in the onset of CHS to oxazolone (Oxa), a well-characterized chemical allergen. In addition, we demonstrate that administration of the rsFcepsilonRIalpha after sensitization successfully prevents murine CHS to Oxa. In a chronic CD model elicited by multiple challenges with low-dose Oxa, application of the rsFcepsilonRIalpha during the course of the challenges showed suppressive effects on CHS to Oxa. Taken together, our data indicate that inhibition of FcepsilonRI-dependent MC activation can suppress allergic CD.

Nck Adapters Are Involved in the Formation of Dorsal Ruffles, Cell Migration, and Rho Signaling Downstream of the Platelet-derived Growth Factor β Receptor

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.

381. Next Generation of Recombinant Adeno-Associated Virus 2 Vectors: Point Mutations in Tyrosine Residues Lead to High-Efficiency Transduction at Lower Vector Doses

381. Next Generation of Recombinant Adeno-Associated Virus 2 Vectors: Point Mutations in Tyrosine Residues Lead to High-Efficiency Transduction at Lower Vector Doses

Amyloid beta protein‐related death‐inducing protein induces G2/M arrest: Implications for neurodegeneration in Alzheimer's disease

Amyloid beta protein (Abeta)-related death-inducing protein (AB-DIP) is a novel Abeta binding protein expressed ubiquitously. Here we demonstrate that overexpression of AB-DIP in SH-SY5Y neuroblastoma cells causes G2/M arrest. By deletion mutant analysis, we have identified the minimal region within AB-DIP required for G2/M arrest. We have also shown that microtubule-interfering agents (MIAs) such as nocodazole, vinblastine, paclitaxel, and vincristine, known to arrest cells at G2/M, also phosphorylate AB-DIP. However, etoposide, which causes genotoxic stress; tunicamycin, an ER stress inducer; and rotenone, which causes mitochondrial damage, fail to phosphorylate AB-DIP, implying that phosphorylation of AB-DIP is specific to microtubule-disruption-induced G2/M arrest. By using different classes of kinase inhibitors, we also demonstrate that a putative tyrosine kinase phosphorylates AB-DIP. Mono- or multisite mutations of tyrosine or serine/threonine residues confirmed that mutation of tyrosine residues but not serine/threonine residues greatly reduces nocodazole-induced phosphorylation of AB-DIP. Furthermore, phosphorylation of AB-DIP can be induced in MCF-7 cells that lack functional p53, suggesting that AB-DIP phosphorylation is independent of p53. Mounting experimental evidence continues to support the role of cell cycle abnormalities in the pathogenesis of Alzheimer's disease, and our results suggest that AB-DIP might provide a mechanistic link between microtubule disruption, mitotic abnormalities, neuronal dysfunction, and death. Therefore, interfering with AB-DIP may have therapeutic applications in conditions such as Alzheimer's disease, in which microtubule disruption and mitotic abnormalities have been suggested to play a pathological role.

Qualitatively differential regulation of T cell activation and apoptosis by T cell receptor zeta chain ITAMs and their tyrosine residues.

The issue of whether three ITAMs in the TCR zeta chain can transmit qualitatively distinct signals or redundantly amplify TCR-mediated activation signals was extensively investigated using stable hCD8-zeta Jurkat transfectants which contain stepwise deletions of each ITAM or mutations of tyrosine residues in each ITAM of TCR zeta chain. The influence of mutations of each tyrosine residue on reduction of the amount and species of tyrosine phosphorylated proteins recruited to zeta chain was quite distinctive, but they were roughly proportional to the number of functionally intact ITAMs. However, the first N-terminal ITAM had a signaling potential to trigger most intracellular signaling events for T cell activation and apoptosis similar to wild-type CD8-zeta, but this level was substantially reduced in the presence of the first and second N-terminal ITAM together. Mutations of tyrosine residues in first and second N-terminal ITAM significantly impaired most signaling events leading to T cell activation and activation-induced cell death, but phosphorylation of mitogen-activated protein kinases (MAPKs) was differentially impaired in each mutant. The mutation of the first tyrosine residue in C-terminal ITAM did not show any impairment in induction of surface antigens and cell death, but rather increased IL-2 secretion and MAPK phosphorylation. Therefore, in this study we demonstrated that the ITAMs and their tyrosine residues of TCR zeta chain can transmit qualitatively differential intracellular signals upon TCR stimulation through distinctive regulation of recruitment of tyrosine phosphorylated proteins to zeta chain and activation of various MAPKs.

The Serum and Glucocorticoid Kinase sgk Increases the Abundance of Epithelial Sodium Channels in the Plasma Membrane of Xenopus Oocytes

The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of beta and gamma subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.