John Overvoorde

RPTPα is essential for NCAM-mediated p59fyn activation and neurite elongation

The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTPα, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton–mediated cross-linking of NCAM and RPTPα in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTPα-deficient brains and is disrupted by dominant-negative RPTPα mutants, demonstrating that RPTPα is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTPα-deficient neurons, and disruption of the NCAM–p59fyn complex in RPTPα-deficient neurons or with dominant-negative RPTPα mutants blocks NCAM-dependent neurite outgrowth, implicating RPTPα as a major phosphatase involved in NCAM-mediated signaling.

Differential Oxidation of Protein-tyrosine Phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPα-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPα-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPα-D1 and RPTPα-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPμ-D1, with a similar conformation as RPTPα-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H2O2 concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

BackgroundDimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.ResultsIn order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.ConclusionsWe demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Zebrafish pten genes have overlapping and non-redundant functions in tumorigenesis and embryonic development

In human cancer, PTEN (Phosphatase and TENsin homolog on chromosome 10, also referred to as MMAC1 and TEP1) is a frequently mutated tumor suppressor gene. We have used the zebrafish as a model to investigate the role of Pten in embryonic development and tumorigenesis. The zebrafish genome encodes two pten genes, ptena and ptenb. Here, we report that both Pten gene products from zebrafish are functional. Target-selected inactivation of ptena and ptenb revealed that Ptena and Ptenb have redundant functions in embryonic development, in that ptena−/− and ptenb−/− mutants did not show embryonic phenotypes. Homozygous single mutants survived as adults and they were viable and fertile. Double homozygous ptena−/−ptenb−/− mutants died at 5 days post fertilization with pleiotropic defects. These defects were rescued by treatment with the phosphatidylinositol-3-kinase inhibitor, LY294002. Double homozygous embryos showed enhanced cellular proliferation. In addition, cell survival was dramatically enhanced in embryos that lack functional Pten upon γ-irradiation. Surprisingly, adult ptenb−/− zebrafish developed ocular tumors later in life, despite the expression of ptena in adult eyes. We conclude that whereas Ptena and Ptenb have redundant functions in embryonic development, they apparently do not have completely overlapping functions later in life. These pten mutant zebrafish represent a unique model to screen for genetic and/or chemical suppressors of Pten loss-of-function.

Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects

SUMMARY Noonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome.

Redox regulation of dimerization of the receptor protein-tyrosine phosphatases RPTPalpha, LAR, RPTPmu and CD45.

Whether dimerization is a general regulatory mechanism of receptor protein‐tyrosine phosphatases (RPTPs) is a subject of debate. Biochemical evidence demonstrates that RPTPα and cluster of differentiation (CD)45 dimerize. Their catalytic activity is regulated by dimerization and structural evidence from RPTPα supports dimerization‐induced inhibition of catalytic activity. The crystal structures of CD45 and leukocyte common antigen related (LAR) indicate that dimerization would result in a steric clash. Here, we investigate dimerization of four RPTPs. We demonstrate that LAR and RPTPμ dimerized constitutively, which is likely to be due to their ectodomains. To investigate the role of the cytoplasmic domain in dimerization we generated RPTPα ectodomain (EDα)/RPTP chimeras and found that – similarly to native RPTPα– oxidation stabilized their dimerization. Limited tryptic proteolysis demonstrated that oxidation induced conformational changes in the cytoplasmic domains of these RPTPs, indicating that the cytoplasmic domains are not rigid structures, but rather that there is flexibility. Moreover, oxidation induced changes in the rotational coupling of dimers of full length EDα/RPTP chimeras in living cells, which were largely dependent on the catalytic cysteine in the membrane‐distal protein‐tyrosine phosphatase domain of RPTPα and LAR. Our results provide new evidence for redox regulation of dimerized RPTPs.

RPTPα and PTPε signaling via Fyn/Yes and RhoA is essential for zebrafish convergence and extension cell movements during gastrulation

Convergence and extension (C&E) cell movements are essential to shape the body axis during vertebrate gastrulation. We have used the zebrafish to assess the role of the receptor protein-tyrosine phosphatases, RPTPalpha and PTPepsilon, in gastrulation cell movements. Both RPTPalpha and PTPepsilon knockdown and ptpra(-/-) embryos show defects in C&E movements. A method was developed to track gastrulation cell movements using confocal microscopy in a quantitative manner and ptpra(-/-) embryos displayed reduced convergence as well as extension speeds. RPTPalpha and PTPepsilon knockdowns cooperated with knockdown of a well known factor in C&E cell movement, non-canonical Wnt11. RPTPalpha and PTPepsilon dephosphorylate and activate Src family kinases in various cell types in vitro and in vivo. We found that Src family kinase phosphorylation was enhanced in ptpra(-/-) embryos, consistent with reduced Src family kinase activity. Importantly, both ptpra(-/-) and RPTPalpha and PTPepsilon knockdown induced C&E defects were rescued by active Fyn and Yes. Moreover, active RhoA rescued the RPTPalpha and PTPepsilon knockdown and ptpra(-/-) induced gastrulation cell movement defects as well. Our results demonstrate that RPTPalpha and PTPepsilon are essential for C&E movements in a signaling pathway parallel to non-canonical Wnts and upstream of Fyn, Yes and RhoA.

The receptor protein-tyrosine phosphatase, Dep1, acts in arterial/venous cell fate decisions in zebrafish development

Dep1 is a transmembrane protein-tyrosine phosphatase (PTP) that is expressed in vascular endothelial cells and has tumor suppressor activity. Mouse models with gene targeted Dep1 either show vascular defects, or do not show any defects at all. We used the zebrafish to investigate the role of Dep1 in early development. The zebrafish genome encodes two highly homologous Dep1 genes, Dep1a and Dep1b. Morpholinos specific for Dep1a and Dep1b induced defects in vasculature, resulting in defective blood circulation. However, Green Fluorescent Protein expression in fli1a::gfp1 transgenic embryos and cdh5 expression, markers of vascular endothelial cells, were normal upon Dep1a- and Dep1b-MO injection. Molecular markers indicated that arterial specification was reduced and venous markers were expanded in Dep1 morphants. Moreover, the Dep1a/Dep1b knockdowns were rescued by inhibition of Phosphatidylinositol-3 kinase (PI3K) and by expression of active Notch and Grl/Hey2. Our results suggest a model in which Dep1 acts upstream in a signaling pathway inhibiting PI3K, resulting in expression of Notch and Grl, thus regulating arterial specification in development.

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.

PZR coordinates Shp2 Noonan and LEOPARD syndrome signaling in zebrafish and mice

ABSTRACT Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, which manifests in congenital heart disease, short stature, and facial dysmorphia. The complexity of Shp2 signaling is exemplified by the observation that LEOPARD syndrome (LS) patients possess inactivating PTPN11 mutations yet exhibit similar symptoms to NS. Here, we identify “protein zero-related” (PZR), a transmembrane glycoprotein that interfaces with the extracellular matrix to promote cell migration, as a major hyper-tyrosyl-phosphorylated protein in mouse and zebrafish models of NS and LS. PZR hyper-tyrosyl phosphorylation is facilitated in a phosphatase-independent manner by enhanced Src recruitment to NS and LS Shp2. In zebrafish, PZR overexpression recapitulated NS and LS phenotypes. PZR was required for zebrafish gastrulation in a manner dependent upon PZR tyrosyl phosphorylation. Hence, we identify PZR as an NS and LS target. Enhanced PZR-mediated membrane recruitment of Shp2 serves as a common mechanism to direct overlapping pathophysiological characteristics of these PTPN11 mutations.