Regulation of the Src kinase-associated phosphoprotein 55 homologue by the protein tyrosine phosphatase PTP-PEST in the control of cell motility.

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

Receptor tyrosine phosphatase sigma (RPTPσ) regulates, p250GAP, a novel substrate that attenuates Rac signaling.

Cumulative evidence supports an important role for RPTPsigma in the development of the nervous system and nerve regeneration. However, the signaling mechanisms regulated by RPTPsigma remain largely unknown and the identification of RPTPsigma substrate(s) and binding partners is essential to understanding its mechanisms of action. We employed a modified yeast-two-hybrid approach, the yeast substrate-trapping system, to identify new substrates and interacting partners of RPTPsigma. The binding proteins RPTPdelta, Liprinalpha4, p130Cas and Trio were found to interact with RPTPsigma in the yeast system independently of tyrosine phosphorylation. Importantly, using the trapping mutant of RPTPsigma we identified p250GAP as a novel substrate and RPTPsigma displayed its phosphatase specificity toward p250GAP in vitro. In the mammalian expression system, the trapping mutant of RPTPsigma recognized p250GAP as its physiological substrate in the presence of active Fyn, suggesting that the interaction of the two proteins is primarily dependent on tyrosine phosphorylation. Furthermore, p250GAP activity increased in the presence of RPTPsigma leading to attenuated Rac activity. Overexpression of p250GAP and RPTPsigma inhibited axonal outgrowth in differentiated PC12 cells. Cumulative evidence implicates that RPTPsigma modulates the actin cytoskeleton by regulating Rac GTPase activity through p250GAP. Taken together, our results demonstrate for the first time that RPTPsigma modulates Rac dependent activity through regulating a novel substrate, p250GAP.

Corticospinal tract regeneration after spinal cord injury in receptor protein tyrosine phosphatase sigma deficient mice.

Receptor protein tyrosine phosphatase sigma (RPTPsigma) plays a role in inhibiting axon growth during development. It has also been shown to slow axon regeneration after peripheral nerve injury and inhibit axon regeneration in the optic nerve. Here, we assessed the ability of the corticospinal tract (CST) axons to regenerate after spinal hemisection and contusion injury in RPTPsigma deficient (RPTPsigma(-/-)) mice. We show that damaged CST fibers in RPTPsigma(-/-) mice regenerate and appear to extend for long distances after a dorsal hemisection or contusion injury of the thoracic spinal cord. In contrast, no long distance axon regeneration of CST fibers is seen after similar lesions in wild-type mice. In vitro experiments indicate that cerebellar granule neurons from RPTPsigma(-/-) mice have reduced sensitivity to the inhibitory effects of chondroitin sulfate proteoglycan (CSPG) substrate, but not myelin, which may contribute to the growth of CST axons across the CSPG-rich glial scar. Our data suggest that RPTPsigma may function to prevent axonal growth after injury in the adult mammalian spinal cord and could be a target for promoting long distance regeneration after spinal cord injury.

Maturation of ureter-bladder connection in mice is controlled by LAR family receptor protein tyrosine phosphatases.

Congenital anomalies affecting the ureter-bladder junction are frequent in newborns and are often associated with other developmental defects. However, the molecular and morphological processes underlying these malformations are still poorly defined. In this study, we identified the leukocyte antigen-related (LAR) family protein tyrosine phosphatase, receptor type, S and F (Ptprs and Ptprf [also known as Lar], respectively), as crucially important for distal ureter maturation and craniofacial morphogenesis in the mouse. Embryos lacking both Ptprs and Ptprf displayed severe urogenital malformations, characterized by hydroureter and ureterocele, and craniofacial defects such as cleft palate, micrognathia, and exencephaly. The detailed analysis of distal ureter maturation, the process by which the ureter is displaced toward its final position in the bladder wall, leads us to propose a revised model of ureter maturation in normal embryos. This process was deficient in embryos lacking Ptprs and Ptprf as a result of a marked reduction in intrinsic programmed cell death, thereby causing urogenital system malformations. In cell culture, Ptprs bound and negatively regulated the phosphorylation and signaling of the Ret receptor tyrosine kinase, whereas Ptprs-induced apoptosis was inhibited by Ret expression. Together, these results suggest that ureter positioning is controlled by the opposing actions of Ret and LAR family phosphatases regulating apoptosis-mediated tissue morphogenesis.

Altered glucose homeostasis in mice lacking the receptor protein tyrosine phosphatase sigma.

Several protein tyrosine phosphatases (PTPs) expressed in insulin sensitive-tissues are proposed to attenuate insulin action and could act as key regulators of the insulin receptor (IR) signaling pathway. Among these PTPs, RPTPsigma is expressed in relatively high levels in insulin-target tissues. We show that RPTPsigma-/- knockout mice have reduced plasma glucose and insulin concentrations in the fasted state compared with their wild-type siblings. The knockout animals were also more sensitive to exogenous insulin as assayed by insulin-tolerance tests. Despite increased whole-body insulin sensitivity, tyrosine phosphorylation of the IR was not increased in muscle of RPTPsigma-/- animals, as would be expected in insulin-sensitive animals. Instead, the levels of IR tyrosine phosphorylation and PI3-kinase activity were reduced in the muscle of knockout animals stimulated with insulin in vivo. However, insulin-stimulated Akt serine phosphorylation was essentially identical between both groups of mice. Accordingly, muscles isolated from RPTPsigma-/- mice did not have a significant increase in glucose uptake in response to insulin, suggesting that RPTPsigma did not play a direct role in this process. Taken together, our results suggest an indirect modulation of the IR signaling pathways by RPTPsigma. Since low dose injection of growth hormone (GH) normalized the response to exogenous insulin in RPTPsigma-/- mice, we propose that the insulin hypersensitivity observed in RPTPsigma-/- mice is secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency.

Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta.

The leukocyte common antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs), LAR, RPTP-sigma, and RPTP-delta, regulate neuroendocrine development, axonal regeneration, and hippocampal long-term potentiation in mammals. In Drosophila, RPTPs are required for appropriate axon targeting during embryonic development. In contrast, deletion of any one of the three LAR-RPTP family members in mammals does not result in gross axon targeting defects. Both RPTP-sigma and RPTP-delta are highly expressed in the developing mammalian nervous system, suggesting they might be functionally redundant. To test this hypothesis, we generated RPTP-sigma and RPTP-delta (RPTP-sigma/delta) double-mutant mice. Although embryonic day 18.5 RPTP-sigma and RPTP-delta single-mutant embryos were viable, RPTP-sigma/delta double mutants were paralyzed, were never observed to draw a breath, and died shortly after cesarean section. RPTP-sigma/delta double mutants exhibit severe muscle dysgenesis and severe loss of motoneurons in the spinal cord. Detailed analysis of the projections of phrenic nerves in RPTP-sigma/delta double mutants indicated that these motoneuron axons emerge normally from the cervical spinal cord, but stall on reaching the diaphragm. Our results demonstrate that RPTP-sigma and RPTP-delta complement each other functionally during mammalian development, and reveal an essential contribution of RPTP-sigma and RPTP-delta to appropriate motoneuron axon targeting during mammalian axonogenesis.

Functional significance of the LAR receptor protein tyrosine phosphatase family in development and diseases.

The protein tyrosine phosphatases (PTPs) have emerged as critical players in diverse cellular functions. The focus of this review is the leukocyte common antigen-related (LAR) subfamily of receptor PTPs (RPTPs). This subfamily is composed of three vertebrate homologs, LAR, RPTP-sigma, and RPTP-delta, as well as few invertebrates orthologs such as Dlar. LAR-RPTPs have a predominant function in nervous system development that is conserved throughout evolution. Proteolytic cleavage of LAR-RPTP proproteins results in the noncovalent association of an extracellular domain resembling cell adhesion molecules and intracellular tandem PTPs domains, which is likely regulated via dimerization. Their receptor-like structures allow them to sense the extracellular environment and transduce signals intracellularly via their cytosolic PTP domains. Although many interacting partners of the LAR-RPTPs have been identified and suggest a role for the LAR-RPTPs in actin remodeling, very little is known about the mechanisms of action of RPTPs. LAR-RPTPs recently raised a lot of interest when they were shown to regulate neurite growth and nerve regeneration in transgenic animal models. In addition, LAR-RPTPs have also been implicated in metabolic regulation and cancer. This RPTP subfamily is likely to become important as drug targets in these various human pathologies, but further understanding of their complex signal transduction cascades will be required.