The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTP, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTP wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTP wedge Tat peptide had no effect on PC12 cells but blocked the PTP-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTP substrate, whereas LAR wedge peptide had no effect. The survival-and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/ TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function.Within intracellular signaling networks, protein-tyrosine kinases (PTKs) 3 can be counterbalanced by protein-tyrosine phosphatases (PTPs) (1-4). Trk-family neurotrophin PTK receptors undergo ligandinduced tyrosine transphosphorylation and downstream activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase/ AKT signaling pathways (5, 6). A fundamental area of Trk signaling that remains to be investigated is the identification of the PTPs that directly or indirectly associate with Trk receptors and regulate their signaling. The leukocyte common antigen-related (LAR) receptor is a prototypical receptor PTP containing tandem catalytic domains (D1 and D2) in its cytoplasmic region with D1, constituting the primary catalytic site, and D2, conferring regulatory function (7). LAR is expressed by neurons and regulates neuronal survival and neurite outgrowth (2, 8 -14). The physiological ligand(s) for LAR in mammalian systems is unknown, although a LAR ectodomain isoform binds LAR homophilically and promotes neurite outgrowth (15). The LAR enzymatic substrates within neurons remain to be established. Several lines of evidence point to LAR as a candidate PTP modulating Trk phosphorylation. First, LAR and Trk are co-expresse...

The receptor protein-tyrosine phosphatase PTP is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTP contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTP contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTP make it an attractive molecule to transduce signals upon cell-cell contact. PTP has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTP signaling pathway utilized to regulate these events. To aid in the further characterization of PTP signaling pathways, we used a series of GST-PTP fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTP-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTP-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTP, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTP and IQGAP1. These data indicate PTP may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTP signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTP-mediated neurite outgrowth.Reversible protein-tyrosine phosphorylation is a primary mode of regulation for several cellular functions including growth, differentiation, adhesion, and protein trafficking. The overall tyrosine phosphorylation state of a protein is regulated by protein-tyrosine kinases (1) and protein-tyrosine phosphatases (2). Similar to the family of protein-tyrosine kinases, the family of protein-tyrosine phosphatases includes both non-receptor and receptor type enzymes. Unlike the tyrosine kinases, however, few substrates and downstream signaling molecules of tyrosine phosphatases have been identified. This manuscript focuses on the receptor protein-tyrosine phosphatase (RPTP), 2 PTP, and the identification of novel PTP-interacting proteins to aid in the elucidation of the PTP signaling pathway. The extracellular domain of PTP contains sequence motifs similar to those in cell adhesion molecules including: a MAM domain, an immunoglobulin (Ig) domain, and four fibronectin type-III repeats (3). The MAM domain (Meprins, A5, PTP Mu) is a sequence motif that is suggested to play a role in protein dimerization (4). Ig domains are disulfide-bonded structures found in many cell surface proteins and have been shown to mediate homophilic and heterophilic binding between cell adhesion molecules. Fibronectin type-III motifs, originally identified in the extracellular matrix protein fibronectin, are present in many cell adhesion molecules. PTP has been show...

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