Gene name - Ptpmeg
Cytological map position-61C1-61C1
Function - signaling
Symbol - Ptpmeg
FlyBase ID: FBgn0261985
Genetic map position - 3L
Classification - FERM, PDZ and PTP domains
Cellular location - cytoplasmic
Ptpmeg is a cytoplasmic tyrosine phosphatase containing FERM and PDZ domains. Drosophila Ptpmeg and its vertebrate homologs PTPN3 and PTPN4 are expressed in the nervous system, but their developmental functions have been unknown. This study found that ptpmeg is involved in neuronal circuit formation in the Drosophila central brain, regulating both the establishment and the stabilization of axonal projection patterns. In ptpmeg mutants, mushroom body (MB) axon branches are elaborated normally, but the projection patterns in many hemispheres become progressively abnormal as the animals reach adulthood. The two branches of MB α/ß neurons are affected by ptpmeg in different ways; ptpmeg activity inhibits α lobe branch retraction while preventing ß lobe branch overextension. The phosphatase activity of Ptpmeg is essential for both α and ß lobe formation, but the FERM domain is required only for preventing α lobe retraction, suggesting that Ptpmeg has distinct roles in regulating the formation of α and ß lobes. ptpmeg is also important for the formation of the ellipsoid body (EB), where it influences the pathfinding of EB axons. ptpmeg function in neurons is sufficient to support normal wiring of both the EB and MB. However, ptpmeg does not act in either MB or EB neurons, implicating ptpmeg in the regulation of cell-cell signaling events that control the behavior of these axons (Whited, 2007).
Neuronal wiring patterns are crucial determinants of brain function. During development, axons navigate to reach their appropriate targets in response to guidance information in their environment. Once established, axonal projection patterns must be appropriately refined and maintained as the nervous system matures and ages. The maintenance of axonal projections is an active process important for nervous system development, with the selective retention of axonal input sculpting patterns of neuronal connectivity. Disruptions in the maintenance of axonal projections are also implicated in human disease, and axonal atrophy is observed in several common neurological disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Understanding the molecular mechanisms that control the establishment and maintenance of neuronal connectivity patterns is therefore critical for understanding how the brain's wiring pattern arises during development and how it is maintained in healthy adults (Whited, 2007).
Cell-cell communication is critical for establishing and maintaining neuronal wiring patterns. The initial pathfinding of axons is modulated by extracellular guidance cues that bind guidance receptors on the axon surface and act to repel or attract the growth cone at the axon tip. During the maturation of the nervous system, patterns of axon branch retention and pruning are also strongly influenced by environmental signals. Some signals that control the maintenance of neuronal wiring act systemically, as in Drosophila where the hormone ecdysone modulates neuronal remodeling throughout the nervous system. Other signals that affect the maintenance of neuronal wiring act more locally, as in the mammalian forebrain where semaphorin proteins trigger the pruning of axon branches (Whited, 2007).
Like pruning, the long-term retention of axon branches is an active process involving cell-cell communication. In the Drosophila mushroom bodies, the maintenance of axon branches requires the inhibition of axon branch retraction by RhoGAP, a negative regulator of Rho and related pathways are proposed to act in mice, where focal adhesion kinase negatively regulates axon branch stabilization via Rho GTPases. Despite the importance of axon branch maintenance to the function of neural circuits, little is known about the molecular mechanisms of long-term axon branch maintenance (Whited, 2007).
Tyrosine phosphatases have important roles in the establishment of neuronal connectivity. In Drosophila, the neuronally expressed receptor tyrosine phosphatases LAR, PTP10D, PTP52F, PTP69D, and PTP99A contribute to axon guidance decisions, and LAR regulates synaptogenesis at the neuromuscular junction. In vertebrates, LAR also regulates the formation and maintenance of synapses. Drosophila ptpmeg encodes an evolutionarily conserved cytoplasmic protein tyrosine phosphatase that is characterized by the presence of an N-terminal FERM domain followed by a single PDZ domain. FERM domains are multi-functional protein and lipid binding domains commonly found in membrane-associated signaling and cytoskeletal proteins. PDZ domains are protein-binding motifs often found in scaffolding proteins (Whited, 2007).
Orthologs of Ptpmeg are present in animals from flies to humans. There are two mammalian homologs of Ptpmeg: PTPN3, which acts as a colon cancer tumor suppressor gene in humans (Wang, 2004), and PTPN4. Both PTPN3 and PTPN4, as well as the C. elegans ortholog PTP-1/PTP-FERM, are neuronally expressed (Hironaka, 2000; Sahin, 1995; Takeuchi, 1994; Uchida, 2002), but the developmental functions of these proteins have not been examined. PTPN4 has been detected in post-synaptic density fractions and physically associates with two predominantly post-synaptic proteins, NMDAR2B [also known as glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GRIN2B); gene ID: 14812], a glutamate receptor subunit, and GRID2 (glutamate receptor, ionotropic, delta 2 MG1:95813), a glutamate-receptor related protein (Hironaka, 2000). Both NMDAR2B and GRID2 are important for brain development and function, although the contribution of PTPN4 to their activities is unknown. Among other functions, both NMDAR2B and GRID2 regulate the branching of axons, acting in target cells to control the behavior of innervating axons (Whited, 2007).
This study characterized the function of ptpmeg in Drosophila by analyzing the effects of ptpmeg mutations on neuronal development. ptpmeg was found to be required to stabilize patterns of mushroom body (MB) axon branching as animals reach adulthood and for axon pathfinding in the developing ellipsoid body (EB). Structure-function studies indicate Ptpmeg phosphatase activity is required for normal MB and EB axon patterning, whereas the FERM domain appears specifically required for stabilizing a particular subset of MB axon branches. Together these data substantiate a role for Ptpmeg in the establishment and maintenance of neuronal wiring patterns (Whited, 2007).
This study found that the evolutionarily conserved cytoplasmic tyrosine phosphatase Ptpmeg contributes to the establishment and the maintenance of axonal projections in the Drosophila central brain. ptpmeg is required for the proper establishment of axon projections in the ellipsoid body (EB), where formation of the EB axon ring is not completed in the absence of ptpmeg. ptpmeg is also required for the formation of normal patterns of axonal projections in the adult mushroom body (MB), but in this case ptpmeg is required to stabilize MB axon projection patterns that have already formed. In the MB, ptpmeg promotes the retention of the dorsally directed α and α' axon branches and inhibits the overgrowth of the medially-directed ß and ß' axon branches. The FERM domain of Ptpmeg is required for MB dorsal branch retention, but is dispensable for preventing medial branch overgrowth, suggesting ptpmeg functions via distinct molecular pathways in dorsal and medial MB axon branch stabilization. Members of the Ptpmeg family of tyrosine phosphatase are neuronally expressed in animals from worms to flies to mice. The present work provides the first evidence that a member of the Ptpmeg family is important for neuronal connectivity (Whited, 2007).
The loss of ptpmeg function has different effects on dorsal and medial MB axon branches. In one model, ptpmeg would primarily affect one set of MB axon branches, with the other set of branches affected secondarily. Alternatively, ptpmeg could affect dorsal and medial branches separately. Both structure-function and phenotypic analyses suggest ptpmeg affects dorsal and medial branches separately. The FERM domain of Ptpmeg is not required to stabilize the medial ß lobes, but is essential for stabilizing the dorsal α lobes. Furthermore, in ptpmeg1 animals with fused ß lobes, ~25% of α lobes appeared normal. Similarly, in ptpmeg1 hemispheres with reduced α lobes, ~15% of ß lobes appeared normal. Therefore, ß lobe overextension does not always accompany α lobe reduction in ptpmeg mutants and vice versa. Taken together, these data suggest Ptpmeg affects α and ß lobes separately, acting to inhibit α lobe retraction and ß lobe overextension. Since ptpmeg is not required within MB neurons, this suggests that Ptpmeg acts in cells that communicate to dorsal branches and in cells that communicate to medial branches (Whited, 2007).
The ability of ptpmeg to promote the retention of α and α' axon branches could reflect the inhibition of either axon degeneration or retraction by ptpmeg. Degenerating and retracting axons often exhibit distinct morphologies. For example, in degenerating axons, such as the axons of MB γ neurons that degenerate during Drosophila metamorphosis, the entire axon branch often appears to degenerate simultaneously. However, retracting axons often exhibit preferential reductions in thickness at the distal end of the axon branch, with small dots of axonal material left behind. The withdrawal of α lobe axons in ptpmeg mutants does not resemble previously characterized axon branch degeneration, but rather resembles axon retraction, as α lobe reduction appears to proceed in a distal to proximal fashion. In addition, the distal tip of the withdrawing branch is often pointed and small dots of axonal material often lie nearby. Similar morphologies are also associated with branch retraction in other systems, and so it is proposed that Ptpmeg inhibits axon retraction pathways in the dorsal lobes (Whited, 2007).
Previous evidence indicates that the persistent inhibition of axon retraction pathways is important for long-term maintenance of α and α' dorsal lobes. Reductions in the expression of Drosophila RhoGAP, which is proposed to act by inhibiting a Rho-dependent axon retraction pathway, cause dorsal lobe retraction resembling that in ptpmeg mutants. However, there are significant differences between ptpmeg and RhoGAP mutant phenotypes. RhoGAP inhibition causes medial lobe retraction whereas ptpmeg mutation cause medial lobe overextension. Furthermore, defects are detected earlier in RhoGAP than in ptpmeg mutants, with ~50% of RhoGAP RNAi hemispheres exhibiting dorsal lobe reduction by 18 hours PPF, increasing to ~95% by 36 hours PPF. Finally, RhoGAP is required in the MB neurons, but ptpmeg is not. Therefore, it is suggested that Ptpmeg participates in additional mechanisms that maintain mushroom body axon branches (Whited, 2007).
In contrast to the retraction of dorsal MB lobes, there is limited precedent for mechanisms that underlie overextension of medial lobe MB axons across the midline. Although several mutants with MB midline crossing defects have been described, a detailed time-course that could distinguish pathfinding defects from later onset defects has been reported only for fmr1 mutants, defective in the Drosophila homolog of the fragile X mental retardation gene. In fmr1 mutants, α/ß axons extend branches across the midline by 24 hours PPF and medial lobe fusion appears complete by 48 hours PPF, consistent with a defect in initial outgrowth. By contrast, ptpmeg1 mutants exhibit no midline crossing defects at 48 hours PPF, suggesting most ß lobe axons initially terminate extension, but reinitiate growth at later stages to cross the midline. Alternatively, midline crossing could be restricted to just the subset of ß axon branches that arrive after 48 hours PPF and might reflect the failure of these axons to stop their initial extension. However, the severity of MB fusion observed in many ptpmeg adults suggests a large proportion of ß lobe axons contribute to the phenotype, consistent with the former explanation (Whited, 2007).
How Ptpmeg might influence 'maintenance' of axon projections after initial extension remains to be determined. MB neurons show no evidence of degeneration in ptpmeg mutants; both their cell body and dendritic regions appear normal. One possible source of MB defects is that Ptpmeg could act in synaptic partners of MB neurons and affect axon target recognition or synaptogenesis. A potentially similar scenario has been observed in the cerebellum of mice mutant for GRID2, a PTPN4-interacting protein (Takeuchi, 2005). Alternatively, Ptpmeg could control the production of structures or signals that influence MB axon behavior more indirectly. Identifying the critical cell populations and molecular pathways through which Ptpmeg modulates MB axon behavior will help determine the basis of these defects. Interestingly, ectopic expression of Ptpmeg in the eye and wing antagonizes the effects of insulin receptor signaling; however, such interactions have been observed only in the context of misexpression (Whited, 2007).
ptpmeg is critical for formation of the EB, a higher order brain region implicated in the control of locomotion. The EB contains axons that travel to the midline and extend ventrally to form a complete ring. In ptpmeg mutants, the EB axons fail to fully extend ventrally, leaving a ventral opening in the EB. These defects appear to result from a defect in EB axon pathfinding rather than axon maintenance. In contrast to the MB, which formed normally but became increasingly abnormal with time, the EB axon ring never completely formed and the defect did not become more severe with time. Similar defects in EB formation have been observed in other central complex mutants, including ciboulot, which encodes a regulator of actin dynamics. In ciboulot mutants, the defect in EB ring closure was proposed to result from a failure of EB axon extension caused by a defect in actin assembly in the EB axon. Since ptpmeg is not required in the EB neurons, ptpmeg likely affects the production of a structure or signal that influences the ventral extension of EB axons, rather than interacting with ciboulot directly. Interestingly, Ptpmeg is expressed on fibers that cross the midline near the developing central complex, which could potentially affect EB axon pathfinding (Whited, 2007).
The ventral region of the EB lies adjacent to the ß and ß' lobes of the MBs, raising the possibility that EB and MB defects are interrelated. This is thought unlikely since expression of a wild-type Ptpmeg cDNA in a ptpmeg mutant created many animals in which the EB ring was complete, but the medial lobes remained defective. Thus, restoration of the EB ring did not eliminate medial lobe defects and the presence of medial lobe defects were not always accompanied by EB ring defects, suggesting these defects can arise separately during development (Whited, 2007).
In addition to its phosphatase domains, Ptpmeg also contains FERM and PDZ domains, protein interaction motifs that could facilitate the assembly of Ptpmeg into signaling complexes and the binding of substrates. This analysis indicates that the ability of Ptpmeg to bind and dephosphorylate substrates is essential for the function of Ptpmeg, and that the FERM and PDZ domains also contribute to Ptpmeg function. Complete elimination of the FERM domain disrupts the ability of Ptpmeg to prevent α lobe retraction, while other activities supported by Ptpmeg appear largely normal. In the case of the PDZ domain, mutation of conserved residues in the GLGF motif partially reduced the ability of Ptpmeg to support MB formation, but had not effect on EB development. Given the partial effects of the FERM and PDZ mutations on Ptpmeg function, it will be of interest to perform further mutational analyses of Ptpmeg to determine whether the FERM and PDZ domains might have redundant roles or whether the phosphatase domain can perform many of the major functions of Ptpmeg by itself (Whited, 2007).
The presence of PDZ and FERM domains in Ptpmeg raised the possibility that Ptpmeg could act as a scaffolding protein. In the mouse brain, the Ptpmeg homolog PTPN4 binds the glutamate receptor subunit NMDAR2B and the glutamate-receptor related protein GRID2 (GluRΔ2) (Hironaka, 2000), indicating Ptpmeg family members can interact with synaptic receptors. Several PDZ domain containing proteins are important modulators of receptor complex localization and activity at the growth cone tip and synapse, while other PDZ domain proteins regulate neurite morphogenesis by acting more proximal to the cell body through the control of receptor trafficking. Ptpmeg is strongly expressed on fibers in the developing and adult brain, but that synapse-rich neuropil regions of the central brain are largely devoid of Ptpmeg. When examined specifically within EB neurons, Ptpmeg expression is restricted to the cell body and the regions of the neurite proximal to the cell body and is not present on axons. Such localization of Ptpmeg to axonal regions near the cell body and its absence from synaptic regions suggest Ptpmeg could act in cell body-proximal regions to influence neurite behavior (Whited, 2007).
These studies demonstrate a role for Ptpmeg in the stabilization of neuronal connectivity patterns in the fly mushroom body. As the mushroom bodies are critical for olfactory learning and memory, molecular pathways that can elicit structural changes in mushroom body axons, such as the pathways in which Ptpmeg participates, are interesting candidates for mediating structural plasticity in this region. More generally, this work shows that Ptpmeg activity is necessary to prevent a progressive loss of the fly's normal wiring pattern as it matures, inhibiting distal-to-proximal retraction of dorsal lobe MB axon branches and inhibiting delayed overextension of medial lobe MB axon branches. Progressive distal-to-proximal disruptions in axonal branching are commonly observed in CNS neurodegenerative diseases such as Alzheimer's and Parkinson's as well as neuropathies associated with diabetes, alcoholism and AIDS. Understanding the kinds of genetic lesions that can destabilize axon branches and the mechanisms that modulate axon branch maintenance could provide useful insights into the mechanisms that contribute to neurological disorders in humans (Whited, 2007).
Three methuselah genes are carried within the intronic structure of Ptpmeg.
The Ptpmeg subfamily of tyrosine phosphatases is characterized by the presence of FERM, PDZ and PTP domains (Whited, 2007).
A gene encoding a protein tyrosine phosphatase (PTP) contains sequence homology to protein 4.1, designated PTPMEG, has been cloned. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. Recombinant protein was prepared in Sf9 and COS-7 cells to further characterize it. The protein was phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation were all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains. The recombinant protein was cleaved by trypsin and calpain in this region and thereby activated 4-8-fold as assayed using Raytide as substrate. The protein was immunoprecipitated from human platelets with both amino- and carboxyl-terminal antipeptide antibodies to assess the state of the enzyme in these cells. The full-length molecule was found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contained proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocked the agonist-induced proteolysis (Gu, 1996a).
Stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form were established in which the active site cysteine is mutated to serine (PTPMEGCS). Both endogenous and recombinant enzymes were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts for only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, it was found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix (Gu, 1996b).
Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning. Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade. To study molecular mechanisms underlying GluRdelta2-dependent motor learning, yeast two-hybrid screening was employed to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG. PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain. In situ hybridization analysis showed that the PTPMEG mRNA is enriched in mouse thalamus and Purkinje cells. PTPMEG interacts with GluRdelta2 as well as with N-methyl-D-aspartate receptor GluRepsilon1 in cultured cells and in brain. PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain. Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner. Thus, it is concluded that PTPMEG associates directly with GluRdelta2 and GluRepsilon1. Moreover, the data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation (Hironaka, 2000).
Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, a novel mouse protein tyrosine phosphatase (PTP) was identified. This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. Expression of this PTP is highly enriched in the testis in mouse and human and has been termed 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis (Park, 2000).
PTP-FERM is a protein tyrosine phosphatase (PTP) of Caenorhabditis elegans containing a FERM domain and a PDZ domain. This study reports the characterization of PTP-FERM and the essential role of its FERM domain in the localization of PTP-FERM in the worm. There are at least three alternatively spliced PTP-FERM isoforms, all of which contain a band 4.1/FERM domain, a PDZ domain, and a catalytic domain. PTP-FERM possesses phosphatase activity. PTP-FERM is expressed predominantly in neurons in the nerve ring and the ventral nerve cord. PTP-FERM is found in the nerve processes and is enriched in the peri-membrane region. Studies using various deletion mutants revealed that the FERM domain is essential and sufficient for the subcellular localization. These results suggest the essential role of the FERM domain in the function of PTP-FERM in the neurons of C. elegans (Uchida, 2002).
Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation. Human papillomavirus (HPV) E6 oncoproteins have been shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). This study shows that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase (Jing, 2007).
PTPN3 (PTPH1) is a cytoskeletal protein tyrosine phosphatase that has been implicated as a negative regulator of early TCR signal transduction and T cell activation. To determine whether PTPN3 functions as a physiological negative regulator of TCR signaling in primary T cells, gene-trapped and gene-targeted mouse strains were generated that lack expression of catalytically active PTPN3. PTPN3 phosphatase-negative mice were born in expected Mendelian ratios and exhibited normal growth and development. Furthermore, numbers and ratios of T cells in primary and secondary lymphoid organs were unaffected by the PTPN3 mutations and there were no signs of spontaneous T cell activation in the mutant mice with increasing age. TCR-induced signal transduction, cytokine production, and proliferation was normal in PTPN3 phosphatase-negative mice. This was observed using both quiescent T cells and recently stimulated T cells where expression of PTPN3 is substantially up-regulated. It is concluded, therefore, that the phosphatase activity of PTPN3 is dispensable for negative regulation of TCR signal transduction and T cell activation (Bauler, 2007).
Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of (359)serine and (835)serine of 14-3-3beta binding sites to alanine, which slightly reduces the interaction with 14-3-3beta, does not influence the PTPN3 effect. In contrast, mutation of the invariant (842)cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3beta, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3beta, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression (Hsu, 2007).
date revised: 8 July 2007
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