Jan Schepens

Developmental expression of the cell adhesion molecule-like protein tyrosine phosphatases LAR, RPTPδ and RPTPσ in the mouse

Abstract Using RNA in situ hybridization we compared the expression patterns of the cell adhesion molecule-like receptor-type protein tyrosine phosphatases LAR, RPTP δ and RPTP σ during mouse development. We found that LAR is expressed in basal lamina-associated epithelial tissues of (neuro)ectodermal, neural crest/ectomesenchyme and endodermal origin. RPTP σ is found in (neuro)ectodermal, neural crest-derived systems and in mesoderm-derived tissues. The expression pattern of RPTP σ largely parallels that of RPTP σ , in concordance with their proposed evolutionary history ( Schaapveld et al., 1995 ).

The neuronal nitric oxide synthase PDZ motif binds to -G(D,E)XV* carboxyterminal sequences

PDZ motifs are small protein–protein interaction modules that are thought to play a role in the clustering of submembranous signalling molecules. The specificity and functional consequences of their associative actions is still largely unknown. Using two‐hybrid methodology we here demonstrate that the PDZ motif of neuronal nitric oxide synthase (nNOS) can mediate the binding to several other proteins in brain. Peptide library screening showed that proteins bearing a carboxy‐terminal G(D,E)XV* sequence are preferred targets for the nNOS amino‐terminal PDZ motif. Potential nNOS targets include a melanoma‐associated antigen, cyclophilins and the α1C‐adrenergic receptor.

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.

Protein tyrosine phosphatases in glioma biology

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 (RPTPδ, DEP1, RPTPμ, RPTPζ) 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.

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.

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-2β, 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-2β 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-2β 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 RPTPα and RPTPε, and down-regulate RPTPα 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-2β.

The mouse Ptprr gene encodes two protein tyrosine phosphatases, PTP-SL and PTPBR7, that display distinct patterns of expression during neural development.

The protein tyrosine phosphatases PTP‐SL and PTPBR7 differ only in the length of their N‐terminal domain. We show here that PTP‐SL and PTPBR7 are isoforms derived from a single gene (Ptprr) through developmentally regulated use of alternative promoters. Isoform‐specific reverse transcriptase‐polymer chain reaction (RT‐PCR) and RNA in situ hybridization experiments reveal that PTPBR7 is expressed during early embryogenesis in spinal ganglia cells as well as in developing Purkinje cells. Post‐natally, PTPBR7 is expressed in various regions of the adult mouse brain, but expression in Purkinje cells has ceased and is replaced by the PTP‐SL‐specific transcript. In transient transfection experiments it is confirmed that PTPBR7 is a type I transmembrane protein tyrosine phosphatase (PTPase). PTP‐SL, however, appears to be a cytosolic membrane‐associated PTPase that is located at perinuclear vesicular structures that partly belong to the endosomal compartment. Thus, during maturation of Purkinje cells, a gene‐promoter switch results in the replacement of a receptor‐type PTPase by a cytosolic vesicle‐associated isoform.

Isolation and characterization of alphoid DNA sequences specific for the pericentric regions of chromosomes 4, 5, 9, and 19

We have cloned and characterized two distinct types of alphoid DNA elements. Probe pG-Xba 11/340 was obtained by random cloning of human satellite DNA and contains two basic units with overall 88% homology to the 171-bp consensus alphoid sequence. pG-Xba 11/340-like elements are represented about 2,000-4,000 times in the haploid genome and, by in situ hybridization, are found exclusively at the primary constrictions of chromosomes 4 and 9. Probe pG-A16 was cloned from a chromosome 19-specific cosmid library and represents a 2.25-kb higher-order DNA element which is present at roughly 75-150 copies per haploid genome and which hybridizes to the centromeres of chromosomes 5 and 19. Using the pG-A16 probe, further genetic and physical dissection of the central area of chromosome 19 can be envisaged.

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

BackgroundRegulation 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.ResultsLTD 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.ConclusionsOur 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.

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

Abstract 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.

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

The use of alternative splice sites, promoters and translation start sites considerably adds to the complexity of organisms. Four mouse cDNAs (PTPBR7, PTP‐SL, PTPPBSγ+ and PTPPBSγ−) 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 PTPPBSγ‐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 PTPPBSγ 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.