Publications by authors named "Jan Schepens"

17 Publications

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Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus.

Philos Trans R Soc Lond B Biol Sci 2019 08 1;374(1779):20180225. Epub 2019 Jul 1.

Department of Cell Biology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Directional cell migration in dense three-dimensional (3D) environments critically depends upon shape adaptation and is impeded depending on the size and rigidity of the nucleus. Accordingly, the nucleus is primarily understood as a physical obstacle; however, its pro-migratory functions by stepwise deformation and reshaping remain unclear. Using atomic force spectroscopy, time-lapse fluorescence microscopy and shape change analysis tools, we determined the nuclear size, deformability, morphology and shape change of HT1080 fibrosarcoma cells expressing the Fucci cell cycle indicator or being pre-treated with chromatin-decondensating agent TSA. We show oscillating peak accelerations during migration through 3D collagen matrices and microdevices that occur during shape reversion of deformed nuclei (recoil), and increase with confinement. During G1 cell-cycle phase, nucleus stiffness was increased and yielded further increased speed fluctuations together with sustained cell migration rates in confinement when compared to interphase populations or to periods of intrinsic nuclear softening in the S/G2 cell-cycle phase. Likewise, nuclear softening by pharmacological chromatin decondensation or after lamin A/C depletion reduced peak oscillations in confinement. In conclusion, deformation and recoil of the stiff nucleus contributes to saltatory locomotion in dense tissues. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
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http://dx.doi.org/10.1098/rstb.2018.0225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627020PMC
August 2019

Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma.

Acta Neuropathol Commun 2016 09 1;4(1):96. Epub 2016 Sep 1.

Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 26, 6525, GA, Nijmegen, The Netherlands.

The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy.
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http://dx.doi.org/10.1186/s40478-016-0372-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009684PMC
September 2016

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

PLoS One 2016 22;11(8):e0160972. Epub 2016 Aug 22.

Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160972PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993582PMC
July 2017

Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

Methods Mol Biol 2016 ;1447:79-93

Biocruces Health Research Institute, Pza Cruces s/n, 48903, Barakaldo, Spain.

In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest.
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http://dx.doi.org/10.1007/978-1-4939-3746-2_5DOI Listing
January 2018

Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma.

Sci Rep 2016 07 27;6:30486. Epub 2016 Jul 27.

Department of Pathology, Radboudumc, Nijmegen, The Netherlands.

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1, and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP(+) to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1(R314C), which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1(R314C) lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP(+), and differs from the IDH1(R132) mutants in that it does not produce D-2-HG. Because IDH1(R314C) is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.
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http://dx.doi.org/10.1038/srep30486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962051PMC
July 2016

Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression.

Mol Brain 2015 Jan 9;8. Epub 2015 Jan 9.

Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, Nijmegen, 6500, HB, The Netherlands.

Background: Regulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop.

Results: LTD in the cerebellum of Ptprr (-/-) mice is strongly impeded, in vitro and in vivo. Comparison of basal phospho-MAPK levels between wild-type and PTPRR deficient cerebellar slices revealed increased levels in mutants. This high basal phospho-MAPK level attenuated further increases in phospho-MAPK during chemical induction of LTD, essentially disrupting the positive feedback loop and preventing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation and endocytosis.

Conclusions: Our findings indicate an important role for PTPRR in maintaining low basal MAPK activity in Purkinje cells. This creates an optimal 'window' to boost MAPK activity following signals that induce LTD, which can then propagate through feed-forward signals to cause AMPAR internalization and LTD.
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http://dx.doi.org/10.1186/s13041-014-0092-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304614PMC
January 2015

PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

Methods 2015 May 22;77-78:147-56. Epub 2014 Oct 22.

Centro de Investigación Príncipe Felipe, Valencia, Spain; BioCruces Health Research Institute, Barakaldo, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. Electronic address:

Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from scaffolding and regulatory proteins. Here, we review the current knowledge on PTEN-PDZ domain interactions and tumor suppressor networks, describe methodology suitable to analyze these interactions, and report the binding of PTEN and the PDZ domain-containing protein tyrosine phosphatase PTPN13. Yeast two-hybrid and GST pull-down analyses showed that PTEN binds to PDZ2/PTPN13 domain in a manner that depends on the specific PTPN13 PDZ domain arrangement involving the interdomain region between PDZ1 and PDZ2. Furthermore, a specific binding profile of PTEN to PDZ2/PTPN13 domain was observed by mutational analysis of the PTEN PDZ-BM. Our results disclose a PDZ-mediated physical interaction of PTEN and PTPN13 with potential relevance in tumor suppression and cell homeostasis.
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http://dx.doi.org/10.1016/j.ymeth.2014.10.017DOI Listing
May 2015

Intracellular and extracellular domains of protein tyrosine phosphatase PTPRZ-B differentially regulate glioma cell growth and motility.

Oncotarget 2014 Sep;5(18):8690-702

Department of Cell Biology , Radboud University Medical Center, Nijmegen, The Netherlands.

Gliomas are primary brain tumors for which surgical resection and radiotherapy is difficult because of the diffuse infiltrative growth of the tumor into the brain parenchyma. For development of alternative, drug-based, therapies more insight in the molecular processes that steer this typical growth and morphodynamic behavior of glioma cells is needed. Protein tyrosine phosphatase PTPRZ-B is a transmembrane signaling molecule that is found to be strongly up-regulated in glioma specimens. We assessed the contribution of PTPRZ-B protein domains to tumor cell growth and migration, via lentiviral knock-down and over-expression using clinically relevant glioma xenografts and their derived cell models. PTPRZ-B knock-down resulted in reduced migration and proliferation of glioma cells in vitro and also inhibited tumor growth in vivo. Interestingly, expression of only the PTPRZ-B extracellular segment was sufficient to rescue the in vitro migratory phenotype that resulted from PTPRZ-B knock-down. In contrast, PTPRZ-B knock-down effects on proliferation could be reverted only after re-expression of PTPRZ-B variants that contained its C-terminal PDZ binding domain. Thus, distinct domains of PTPRZ-B are differentially required for migration and proliferation of glioma cells, respectively. PTPRZ-B signaling pathways therefore represent attractive therapeutic entry points to combat these tumors.
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http://dx.doi.org/10.18632/oncotarget.2366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226714PMC
September 2014

Protein tyrosine phosphatases in glioma biology.

Acta Neuropathol 2010 Feb 21;119(2):157-75. Epub 2009 Nov 21.

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands.

Gliomas are a diverse group of brain tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. In combination with their refractive nature to chemotherapy this makes it almost impossible to cure patients using combinations of conventional therapeutic strategies. The drastically increased knowledge about the molecular underpinnings of gliomas during the last decade has elicited high expectations for a more rational and effective therapy for these tumors. Most studies on the molecular pathways involved in glioma biology thus far had a strong focus on growth factor receptor protein tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. Except for the tumor suppressor PTEN, much less attention has been paid to the PTK counterparts, the protein tyrosine phosphatase (PTP) superfamily, in gliomas. PTPs are instrumental in the reversible phosphorylation of tyrosine residues and have emerged as important regulators of signaling pathways that are linked to various developmental and disease-related processes. Here, we provide an overview of the current knowledge on PTP involvement in gliomagenesis. So far, the data point to the potential implication of receptor-type (RPTPdelta, DEP1, RPTPmicro, RPTPzeta) and intracellular (PTP1B, TCPTP, SHP2, PTPN13) classical PTPs, dual-specific PTPs (MKP-1, VHP, PRL-3, KAP, PTEN) and the CDC25B and CDC25C PTPs in glioma biology. Like PTKs, these PTPs may represent promising targets for the development of novel diagnostic and therapeutic strategies in the treatment of high-grade gliomas.
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http://dx.doi.org/10.1007/s00401-009-0614-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808538PMC
February 2010

Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7 and PTP-SL attenuates enzymatic activity.

Biochim Biophys Acta 2008 Feb 17;1783(2):275-86. Epub 2007 Nov 17.

Department of Cell Biology, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.

Dimerisation of receptor-type protein tyrosine phosphatases (RPTPs) represents an appealing mechanism to regulate their enzymatic activity. Studies thus far mostly concern the dimerisation behaviour of RPTPs possessing two tandemly oriented catalytic PTP domains. Mouse gene Ptprr encodes four different protein isoforms (i.e. PTPBR7, PTP-SL and PTPPBSgamma-42/37) that contain a single PTP domain. Using selective membrane permeabilisation we here demonstrate that PTP-SL, like PTPBR7, is a single membrane-spanning RPTP. Furthermore, these two receptor-type PTPs constitutively formed homo- and hetero-meric complexes as witnessed in chemical cross-linking and co-immunoprecipitation experiments, in sharp contrast to the cytosolic PTPPBSgamma-42 and PTPPBSgamma-37 PTPRR isoforms. This multimerisation occurs independently of the PTP domain and requires the transmembrane domain and/or the proximal hydrophobic region. Using overexpression of a PTPBR7 mutant that essentially lacks the intracellular PTP domain-containing segment, a monomer-mimicking state was forced upon full-length PTPBR7 immunoprecipitates. This resulted in a significant increase in the enzymatic activity of the PTPRR PTP domain, which strengthens the notion that multimerisation represents a general mechanism to tone down RPTP catalytic activity.
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http://dx.doi.org/10.1016/j.bbamcr.2007.10.023DOI Listing
February 2008

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1.

J Biol Chem 2005 Nov 7;280(45):37885-94. Epub 2005 Sep 7.

Centro de Investigación Príncipe Felipe, Valencia, Spain.

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.
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http://dx.doi.org/10.1074/jbc.M504366200DOI Listing
November 2005

Characterization of multiple transcripts and isoforms derived from the mouse protein tyrosine phosphatase gene Ptprr.

Genes Cells 2004 Oct;9(10):919-33

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, University of Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.

The use of alternative splice sites, promoters and translation start sites considerably adds to the complexity of organisms. Four mouse cDNAs (PTPBR7, PTP-SL, PTPPBSgamma+ and PTPPBSgamma-) have been cloned that contain different 5' parts but encode identical protein tyrosine phosphatase PTPRR catalytic domains. We investigated the genomic origin and coding potential of these transcripts to elucidate their interrelationship. Mouse gene Ptprr exons were identified within a 260 kbp segment on chromosome 10, revealing PTP-SL- and PTPPBSgamma-specific transcription start sites within introns two and four, respectively, relative to the 14 PTPBR7 exons. Northern and RT-PCR analyses demonstrated differential expression patterns for these promoters. Furthermore, transfection studies and AUG codon mutagenesis demonstrated that in PTP-SL and PTPPBSgamma messengers multiple translation initiation sites are being used. Resulting 72, 60, 42 and 37 kDa PTPRR protein isoforms differ not only in the length of their N-terminal part but also in their subcellular localization, covering all major PTP subtypes; receptor-like, membrane associated and cytosolic. In summary, mouse gene Ptprr gives rise to multiple isoforms through the use of distinct promoters, alternative splicing and differential translation starts. These results set the stage for further investigations on the physiological roles of PTPRR proteins.
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http://dx.doi.org/10.1111/j.1365-2443.2004.00773.xDOI Listing
October 2004

Cloning and characterization of mCRIP2, a mouse LIM-only protein that interacts with PDZ domain IV of PTP-BL.

Genes Cells 2003 Jul;8(7):631-44

Department of Cell Biology, Institute of Cellular Signalling, University Medical Center St. Radboud, University of Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.

Background: In the mouse submembranous protein tyrosine phosphatase PTP-BL five PDZ domains are present in between the N-terminal FERM domain, which directs the protein to the cell cortex, and the C-terminal catalytic phosphatase domain. To understand more on the physical role of PTP-BL in this microenvironment, we started to search for PTP-BL PDZ domain-interacting proteins.

Results: Yeast two-hybrid screening for PTP-BL targets resulted in the identification of a novel mouse LIM-only protein termed CRIP2 that is highly homologous to rat ESP1 and human CRP2 sequences. Mouse CRIP2 has a predicted molecular weight of 23 kD and consists of two LIM domains spaced by 68 amino acids. The fourth PDZ domain of PTP-BL is responsible for the binding of CRIP2 protein. Both PTP-BL and CRIP2 mRNAs display a wide, overlapping tissue distribution. Western blot analysis revealed a more restricted expression pattern for CRIP2 with high expression in lung, heart and brain. CRIP2 protein is localized at cell cortical, actin-rich structures, which is concurrent with the subcellular localization of PTP-BL.

Conclusions: The observed characteristics of the LIM domain-containing adaptor protein CRIP2 are consistent with a potential role of PTP-BL in the dynamics of the cortical actin cytoskeleton.
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http://dx.doi.org/10.1046/j.1365-2443.2003.00660.xDOI Listing
July 2003

Colocalisation of the protein tyrosine phosphatases PTP-SL and PTPBR7 with beta4-adaptin in neuronal cells.

Histochem Cell Biol 2003 Jan 21;119(1):1-13. Epub 2002 Dec 21.

Department of Cell Biology, Nijmegen Center for Molecular Life Sciences, University Medical Center St. Radboud, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands.

The mouse gene Ptprr encodes the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7. These proteins differ in their N-terminal domains, with PTP-SL being a cytosolic, membrane-associated phosphatase and PTPBR7 a type I transmembrane protein. In this study, we further explored the nature of the PTP-SL-associated vesicles in neuronal cells using a panel of organelle markers and noted a comparable subcellular distribution for PTP-SL and the beta4-adaptin subunit of the AP4 complex. PTP-SL, PTPBR7 and beta4-adaptin are localised at the Golgi apparatus and at vesicles throughout the cytoplasm. Immunohistochemical analysis demonstrated that PTP-SL, PTPBR7 and beta4-adaptin are all endogenously expressed in brain. Interestingly, coexpression of PTP-SL and beta4-adaptin leads to an altered subcellular localisation for PTP-SL. Instead of the Golgi and vesicle-type staining pattern, still observable for beta4-adaptin, PTP-SL is now distributed throughout the cytoplasm. Although beta4-adaptin was found to interact with the phosphatase domain of PTP-SL and PTPBR7 in the yeast two-hybrid system, it failed to do so in transfected neuronal cells. Our data suggest that the tyrosine phosphatases PTP-SL and PTPBR7 may be involved in the formation and transport of AP4-coated vesicles or in the dephosphorylation of their transmembrane cargo molecules at or near the Golgi apparatus.
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http://dx.doi.org/10.1007/s00418-002-0489-9DOI Listing
January 2003

Multimerization of the protein-tyrosine phosphatase (PTP)-like insulin-dependent diabetes mellitus autoantigens IA-2 and IA-2beta with receptor PTPs (RPTPs). Inhibition of RPTPalpha enzymatic activity.

J Biol Chem 2002 Dec 2;277(50):48139-45. Epub 2002 Oct 2.

Department of Cell Biology, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, The Netherlands.

Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains, and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2beta, major autoantigens in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2beta are involved in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that IA-2 and IA-2beta are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPalpha and RPTPepsilon, and down-regulate RPTPalpha enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2beta.
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http://dx.doi.org/10.1074/jbc.M208228200DOI Listing
December 2002

Structure, dynamics and binding characteristics of the second PDZ domain of PTP-BL.

J Mol Biol 2002 Mar;316(5):1101-10

NSR-RIM Center, Department of Biophysical Chemistry, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.

The PDZ domains of the protein tyrosine phosphatase PTP-BL mediate interactions by binding to specific amino acid sequences in target proteins. The solution structure of the second PDZ domain of PTP-BL, PDZ2, displays a compact fold with six beta strands and two alpha-helices. A unique feature of this domain compared to the canonical PDZ fold is an extended flexible loop at the base of the binding pocket, termed L1, that folds back onto the protein backbone, a feature that is shared by both the murine and human orthologues. The structure of PDZ2 differs significantly from the orthologous human structure. A comparison of structural quality indicators clearly demonstrates that the PDZ2 ensemble is statistically more reasonable than that of the human orthologue. The analysis of (15)N relaxation data for PDZ2 shows a normal pattern, with more rigid secondary structures and more flexible loop structures. Close to the binding pocket, Leu85 and Thr88 display greater mobility when compared to surrounding residues. Peptide binding studies demonstrated a lack of interaction between murine PDZ2 and the C terminus of the murine Fas/CD95 receptor, suggesting that the Fas/CD95 receptor is not an in vivo target for PDZ2. In addition, PDZ2 specifically binds the C termini of both human Fas/CD95 receptor and the RIL protein, despite RIL containing a non-canonical PDZ-interacting sequence of E-x-V. A model of PDZ2 with the RIL peptide reveals that the PDZ2 binding pocket is able to accommodate the bulkier side-chain of glutamic acid while maintaining crucial protein to peptide hydrogen bond interactions.
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http://dx.doi.org/10.1006/jmbi.2002.5402DOI Listing
March 2002
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