Jeroen den Hertog

Laminin-α4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

Background— Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. Methods and Results— We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-&agr;4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5±3 &mgr;mol/L) and Arg1073X LAMA4 (Kd=1±0.2 &mgr;mol/L) mutants compared with the wild-type LAMA4 protein (Kd=440±20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. Conclusions— This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

Epidermal growth factor activates calcium channels by phospholipase A25-lipoxygenase-mediated leukotriene C4 production

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.

Regulation of receptor protein-tyrosine phosphatase α by oxidative stress

The presence of two protein‐tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane‐distal PTP domain (RPTP‐D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C‐terminus in RPTPα prohibited intermolecular interactions. Interestingly, stress factors such as H2O2, UV and heat shock induced reversible, free radical‐dependent, intermolecular interactions between RPTPα and RPTPα‐SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPα‐SpD2, Cys723, was required for the H2O2 effect on RPTPα. H2O2 induced a rapid, reversible, Cys723‐dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPα‐SpD2 in a single chimeric protein. Importantly, H2O2 treatment stabilized RPTPα dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPα‐D2, leading to stabilization of RPTPα dimers, and thus to inhibition of RPTPα activity.

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

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.

Preferential oxidation of the second phosphatase domain of receptor-like PTP-α revealed by an antibody against oxidized protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) α after treatment of cells with H2O2 were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTPα was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTPα demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.

Receptor-like protein tyrosine phosphatase alpha homodimerizes on the cell surface.

ABSTRACT We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase α (RPTPα) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPς D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPα in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPα dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPα exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPα biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPα homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPα extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPα dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPα. Such RPTPα dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.

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.

Protein tyrosine phosphatases: regulatory mechanisms

Protein‐tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post‐translational modifications affecting intrinsic catalytic activity, ligand binding and dimerization. Here, we review the regulatory mechanisms found to control the classical protein‐tyrosine phosphatases.

Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity.

TGF-β-stimulatedclone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution. Two homologues are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box). Only limited data are available on the function of TSC-22 and its homologues. TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors, and recent data suggest that TSC-22 may play a suppressive role in tumorigenesis. In this paper we show that TSC-22 forms homodimers via its conserved leucine zipper domain. Using a yeast two-hybrid screen, we identified a TSC-22 homologue (THG-1) as heterodimeric partner. Furthermore, we report the presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes. Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNA-binding domain. The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A. Mutational analysis showed that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. Our results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription.