Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca2+, which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the α-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.

Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins).

Staphylococcal enterotoxins (SE) stimulate T cells expressing the appropriate variable region beta chain of (V beta) T-cell receptors and have been implicated in the pathogenesis of several autoimmune diseases. Depending on costimulatory signals, SE induce either proliferation or anergy in T cells. In addition, SE can induce an interleukin-2 (IL-2) nonresponsive state and apoptosis. Here, we show that SE induce dynamic changes in the expression of and signal transduction through the IL-2 receptor (IL-2R) beta and gamma chains (IL-2R beta and IL-2R gamma) in human antigen-specific CD4+ T-cell lines. Thus, after 4 hr of exposure to SEA and SEB, the expression of IL-2R beta was down-regulated, IL-2R gamma was slightly up-regulated, while IL-2R alpha remained largely unaffected. The changes in the composition of IL-2Rs were accompanied by inhibition of IL-2-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription called Stat3 and Stat5. In parallel experiments, IL-2-driven proliferation was inhibited significantly. After 16 hr of exposure to SE, the expression of IL-2R beta remained low, while that of IL2R alpha and IL2R gamma was further up-regulated, and ligand-induced tyrosine phosphorylation of Jak3 and Stat proteins was partly normalized. Yet, IL-2-driven proliferation remained profoundly inhibited, suggesting that signaling events other than Jak3/Stat activation had also been changed following SE stimulation. In conclusion, our data suggest that SE can modulate IL-2R expression and signal transduction involving the Jak/Stat pathway in CD4+ T-cell lines.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Protein oxidation:role in signalling and detection by mass spectrometry

Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while reversible modifications are thought to be relevant in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM, such as oxidation, chlorination or nitration. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to disulfide, glutathionylated or sulfenic acid forms that can be reversed by thiol reductants. Proline hydroxylation in HIF signalling is also quite well characterized, and there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. For some proteins regulated by cysteine oxidation, the residues and molecular mechanism involved have been extensively studied and are well understood, such as the protein tyrosine phosphatase PTP1B and MAP3 kinase ASK1, as well as transcription factor complex Keap1-Nrf2. The advances in understanding of the role oxPTMs in signalling have been facilitated by advances in analytical technology, in particular tandem mass spectrometry techniques. Combinations of peptide sequencing by collisionally induced dissociation and precursor ion scanning or neutral loss to select for specific oxPTMs have proved very useful for identifying oxidatively modified proteins and mapping the sites of oxidation. The development of specific labelling and enrichment procedures for S-nitrosylation or disulfide formation has proved invaluable, and there is ongoing work to establish analogous methods for detection of nitrotyrosine and other modifications.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Neuroprotection and regeneration of adult lesioned retinal ganglion cells by the modulation of fibroblast growth factor-2 and receptor tyrosine phosphatase-sigma

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

In silico analyses reveal common cellular pathways affected by loss of heterozygosity (LOH) events in the lymphomagenesis of Non-Hodgkin’s lymphoma (NHL)

Background The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin’s lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J (PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis. Results Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis (i.e., metabolic pathways). These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of PTPRJ, such as NFATC3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3). Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL. Conclusions The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours.

Genomewide screens in ankylosing spondylitis

Efforts to identify genes other than HLA-B27 in AS have been driven by the strength of the evidence from genetic epidemiology studies indicating that HLA-B27, although a major gene in AS, is clearly not the only significant gene operating. This is the case for both genetic determinants of disease-susceptibility and phenotypic characteristics such as disease severity and associated disease features. In this chapter the genetic epidemiology of AS and the gene-mapping studies performed to date will be reviewed and the future direction of research in this field discussed.