Induction of Neurite Outgrowth through Contactin and Nr-CAM by Extracellular Regions of Glial Receptor Tyrosine Phosphatase β

Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.     

The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase β (RPTPβ)

Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it coordinates signaling between these two receptor-linked signaling systems.     

NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca²⁺ channels, and NCAM clustering at the cell surface results in Ca²⁺ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.     

Loss of Receptor Protein Tyrosine Phosphatase β/ζ (RPTPβ/ζ) Promotes Prostate Cancer Metastasis

Pleiotrophin is a growth factor that induces carcinogenesis. Despite the fact that many published reports focused on the role of pleiotrophin and its receptors, receptor protein tyrosine phosphatase (RPTPβ/ζ), and syndecan-3 during tumor development, no information is available regarding their function in tumor metastasis. To investigate the mechanism through which pleiotrophin regulates tumor metastasis, we used two different prostate carcinoma cell lines, DU145 and PC3, in which the expression of RPTPβ/ζ or syndecan-3 was down-regulated by the RNAi technology. The loss of RPTPβ/ζ expression initiated epithelial-to-mesenchymal transition (EMT) and increased the ability of the cells to migrate and invade. Importantly, the loss of RPTPβ/ζ expression increased metastasis in nude mice in an experimental metastasis assay. We also demonstrate that RPTPβ/ζ counterbalanced the pleiotrophin-mediated syndecan-3 pathway. While the inhibition of syndecan-3 expression inhibited the pleiotrophin-mediated cell migration and attachment through the Src and Fak pathway, the inhibition of RPTPβ/ζ expression increased pleiotrophin-mediated migration and attachment through an interaction with Src and the subsequent activation of a signal transduction pathway involving Fak, Pten, and Erk1/2. Taken together, these results suggest that the loss of RPTPβ/ζ may contribute to the metastasis of prostate cancer cells by inducing EMT and promoting pleiotrophin activity through the syndecan-3 pathway.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.     

Involvement of Receptor-like Protein Tyrosine Phosphatase ζ/RPTPβ and Its Ligand Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM) in Neuronal Migration

Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase ζ (PTPζ)/receptor-like protein tyrosine phosphatase β (RPTPβ) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPζ isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPζ interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-l-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-l-lysine–induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPζ, PTPζ-S, an extracellular secreted form of PTPζ, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPζ, and PTPζ-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPζ are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPζ is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers.     

α-Actinin-4-Mediated FSGS: An Inherited Kidney Disease Caused by an Aggregated and Rapidly Degraded Cytoskeletal Protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in theα-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant α-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant α-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a “knockin” mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant α-actinin-4, mediated, at least in part, by the ubiquitin–proteasome pathway. We correlate these findings with studies of α-actinin-4 expression in human samples. “Knockin” mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 “knockin” and “knockout” mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α_vβ₃ integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α_vβ₃ by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α_vβ₃ and depended on the phosphorylation of β₃ at Tyr⁷⁷³ through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of α_vβ_3, PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α_vβ₃ and RPTPβ/ζ. Positive correlation of cell surface NCL and α_vβ₃ expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α_vβ₃. Collectively, these data suggest that both expression and β₃ integrin phosphorylation at Tyr⁷⁷³ determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.     

Protein phosphatase 2A regulatory subunits affecting plant innate immunity,energy metabolism,and flowering time – joint functions among B'η subfamily members

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic, scaffolding, and regulatory subunit. The regulatory subunits are essential for substrate specificity and localization of the complex and are classified into B/B55, B'', and B” non-related families in higher plants. In Arabidopsis thaliana, the close paralogs B''η, B''θ, B''γ, and B''ζ were further classified into a subfamily of B'' called B''η. Here we present results that consolidate the evidence for a role of the B''η subfamily in regulation of innate immunity, energy metabolism and flowering time. Proliferation of the virulent Pseudomonas syringae in B''θ knockout mutant decreased in comparison with wild type plants. Additionally, B''θ knockout plants were delayed in flowering, and this phenotype was supported by high expression of FLC (FLOWERING LOCUS C). B''ζ knockout seedlings showed growth retardation on sucrose-free medium, indicating a role for B''ζ in energy metabolism. This work provides insight into functions of the B''η subfamily members, highlighting their regulation of shared physiological traits while localizing to distinct cellular compartments.     

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.     

Protein tyrosine phosphatase PTPRT as a regulator of synaptic formation and neuronal development

PTPRT/RPTPρ is the most recently isolated member of the type IIB receptor-type protein tyrosine phosphatase family and its expression is restricted to the nervous system. PTPRT plays a critical role in regulation of synaptic formation and neuronal development. When PTPRT was overexpressed in hippocampal neurons, synaptic formation and dendritic arborization were induced. On the other hand, knockdown of PTPRT decreased neuronal transmission and attenuated neuronal development. PTPRT strengthened neuronal synapses by forming homophilic trans dimers with each other and heterophilic cis complexes with neuronal adhesion molecules. Fyn tyrosine kinase regulated PTPRT activity through phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT. Phosphorylation induced homophilic cis dimerization of PTPRT and resulted in the inhibition of phosphatase activity. BCR-Rac1 GAP and Syntaxin-binding protein were found as new endogenous substrates of PTPRT in rat brain. PTPRT induced polymerization of actin cytoskeleton that determined the morphologies of dendrites and spines by inhibiting BCR-Rac1 GAP activity. Additionally, PTPRT appeared to regulate neurotransmitter release through reinforcement of interactions between Syntaxin-binding protein and Syntaxin, a SNARE protein. In conclusion, PTPRT regulates synaptic function and neuronal development through interactions with neuronal adhesion molecules and the dephosphorylation of synaptic molecules. [BMB Reports 2015; 48(5): 249-255]     

Kinesins in the Arabidopsis genome: A comparative analysis among eukaryotes

Background  

Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPρ gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPρ expression is restricted to the central nervous system.     

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including “ecstasy” (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N⁶-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-³³P]ATP but not from similarly labeled N⁶-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.     

The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a K_D of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.”     

Positive regulation of TNFR1 signaling via SH3 recognition motif

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the “death domain”, it also activates IKKα/β, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.     

The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTPδ, and PTPς, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTPς (PTPς-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTPδ (PTPδ-D2) as an interactor of PTPς-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTPς-D1 and PTPδ-D2, an association which required the presence of the wedge sequence in PTPς-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTPα. This interaction was not reciprocal, as PTPδ-D1 did not bind PTPς-D2. Addition of a glutathione S-transferase (GST)–PTPδ-D2 fusion protein (but not GST alone) to GST–PTPς-D1 led to ~50% inhibition of the catalytic activity of PTPς-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTPς-D1 activity was obtained with coimmunoprecipitated PTPδ-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTPς (PTPς-D2), very similar in sequence to PTPδ-D2, bound poorly to PTPς-D1. PTPδ-D1 and LAR-D1 were also able to bind PTPδ-D2, but more weakly than PTPς-D1, with a binding hierarchy of PTPς-D1>>PTPδ-D1>LAR-D1. These results suggest that association between PTPς-D1 and PTPδ-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.