The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, 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 PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.     

Regulation of receptor protein-tyrosine phosphatase dimerization

Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.     

Coupling factor 1 from Escherichia coli lacking subunits delta and epsilon: preparation and specific binding to depleted membranes, mediated by subunits delta or epsilon

A procedure for the preparation of coupling factor 1 (F1) from Escherichia coli lacking subunits delta and epsilon is described. Using chloroform and dimethyl sulfoxide, we can isolate F1 containing only subunits alpha, beta, and gamma [F1(alpha beta gamma)] directly from membrane vesicles in 10-mg quantities. Pure and active subunits delta and epsilon were prepared from five-subunit F1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After addition of these subunits, F1(alpha beta gamma) is as active in reconstituting ATP-dependent transhydrogenase as five-subunit F1. The ATPase activity of F1 (alpha beta gamma) is inhibited by subunit epsilon in a 1:1 stoichiometry to the same extent (approximately equal to 90%) and with the same affinity (Ki = 0.2-0.8 nM) as reported earlier [Dunn, S.D. (1982) J. Biol. Chem. 257, 7354-7359]. In the presence of either delta or epsilon, F1(alpha beta gamma) binds to F1-depleted membrane vesicles and to liposomes containing the membrane sector (F0) of the ATP synthase to an extent commensurate with the F0 content. The binding ratios epsilon/F1 (alpha beta gamma) and probably also delta/F1 (alpha beta gamma) are close to unity. The specific, delta- or epsilon-deficient F1.F0 complexes presumably formed show ATPase activities sensitive to subunit epsilon but not to dicyclohexylcarbodiimide, and no energy-transfer capabilities. Binding studies at different pH values suggest that F1-F0 interactions in the presence of both subunits delta and epsilon are similar to a combination of those mediated by delta or epsilon alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.     

Adiponectin receptor 1 interacts with both subunits of protein kinase CK2

Adiponectin is an adipose tissue-derived hormone that is involved in the inhibition of metabolic syndrome, protection of hypertension, and suppression of atherosclerosis. Since these effects are not understood in detail, adiponectin signaling has to be clarified for therapeutic applications. Adiponectin activities are mediated by its two receptors adiponectin receptor 1 and adiponectin receptor 2, which consist of seven transmembrane helices. Previous studies revealed the beta subunit of protein kinase CK2 as an interaction partner of the adiponectin receptor 1 N-terminus using a yeast-two-hybrid screen, co-immunoprecipitation, ELISA experiments, and co-localization studies. Inhibition of CK2 activity by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole led to a decrease of ACC phosphorylation and indicates an important role of CK2 in adiponectin signaling. CK2 is characterized as a heterotetramer that consists of two regulatory beta and two catalytic alpha subunits, but a holoenzyme-independent role for both subunits is described as well. Therefore, we analyzed the role of the catalytic subunit in this interaction by co-immunoprecipitation and bimolecular fluorescence complementation studies and found CK2 alpha as an interaction partner of the receptor. Treatment with full-length adiponectin resulted in no dissociation of the catalytic alpha subunit. Consequently, our data suggest an interaction of the adiponectin receptor 1 with the tetrameric complex and identified protein kinase CK2 as a key player in adiponectin signaling.     

Multiple interactions between receptor protein-tyrosine phosphatase (RPTP) alpha and membrane-distal protein-tyrosine phosphatase domains of various RPTPs

Receptor protein-tyrosine phosphatase (RPTP) alpha belongs to the large family of receptor protein-tyrosine phosphatases containing two tandem phosphatase domains. Most of the catalytic activity is retained in the first, membrane-proximal domain (RPTPalpha-D1), and little is known about the function of the second, membrane-distal domain (RPTPalpha-D2). We investigated whether proteins bound to RPTPalpha using the two-hybrid system and found that the second domain of RPTPsigma interacted with the juxtamembrane domain of RPTPalpha. We confirmed this interaction by co-immunoprecipitation experiments. Furthermore, RPTPalpha not only interacted with RPTPsigma-D2 but also with RPTPalpha-D2, LAR-D2, RPTPdelta-D2, and RPTPmu-D2, members of various RPTP subfamilies, although with different affinities. In the yeast two-hybrid system and in glutathione S-transferase pull-down assays, we show that the RPTP-D2s interacted directly with the wedge structure of RPTPalpha-D1 that has been demonstrated to be involved in inactivation of the RPTPalpha-D1/RPTPalpha-D1 homodimer. The interaction was specific because the equivalent wedge structure in LAR was unable to interact with RPTPalpha-D2 or LAR-D2. In vivo, we show that other interaction sites exist as well, including the C terminus of RPTPalpha-D2. The observation that RPTPalpha, but not LAR, bound to multiple RPTP-D2s with varying affinities suggests a specific mechanism of cross-talk between RPTPs that may regulate their biological function.     

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.     

Expression of zebrafish glutamate receptor delta2 in neurons with cerebellum-like wiring

Mammalian glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays key roles in cerebellar plasticity, motor learning, and neural wiring. Here, we isolated cDNA encoding the zebrafish ortholog of mammalian GluRdelta2. We found that in adult zebrafish brain, glurdelta2 mRNA was expressed not only in cerebellar Purkinje cells, but also in the crest cells of the medial octavolateral nucleus (MON) and the type I neurons of the optic tectum. Immunohistochemical analysis revealed that zebrafish GluRdelta2 proteins were selectively localized in the apical dendrites of these neurons. Interestingly, the crest cells of the MON and the type I neurons of the optic tectum receive large numbers of parallel fiber inputs at the apical dendrites and sensory inputs at the proximal or basal dendrites. These results suggest that the expression of zebrafish GluRdelta2 is selective for cerebellum-like neural wiring with large numbers of parallel fiber inputs.     

Fe65 interacts with P2X2 subunits at excitatory synapses and modulates receptor function

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X(2) subunit C-terminal domain as bait we isolated the beta-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X(2) C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X(2) splice variant P2X(2(b)), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X(2) subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X(2) and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X(2) receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X(2) receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.     

Regulation of receptor protein-tyrosine phosphatase alpha by oxidative stress

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPalpha prohibited intermolecular interactions. Interestingly, stress factors such as H(2)O(2), UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPalpha and RPTPalpha-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPalpha-SpD2, Cys723, was required for the H(2)O(2) effect on RPTPalpha. H(2)O(2) induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPalpha-SpD2 in a single chimeric protein. Importantly, H(2)O(2) treatment stabilized RPTPalpha dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPalpha-D2, leading to stabilization of RPTPalpha dimers, and thus to inhibition of RPTPalpha activity.     

Consensus substrate sequence for protein-tyrosine phosphatase receptor type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.     

Metalloproteinase- and gamma-secretase-mediated cleavage of protein-tyrosine phosphatase receptor type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/gamma-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system.     

Redox-regulated rotational coupling of receptor protein-tyrosine phosphatase alpha dimers

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTP alpha revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTP alpha and of constitutively dimeric, active RPTP alpha-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTP alpha-P137C was. All three proteins were expressed on the plasma membrane to a similar extent, and the accessibility of their extracellular domains did not differ as determined by biotinylation studies. Dimerization was required for masking the HA tag, and we identified a region in the N terminus of RPTP alpha that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTP alpha-D2). Here we report that H(2)O(2) treatment of cells induced a change in rotational coupling in RPTP alpha dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys(723) in RPTP alpha-D2, which was required for the conformational change of RPTP alpha-D2 upon H(2)O(2) treatment, was essential for the H(2)O(2)-induced increase in accessibility. These results show for the first time that a conformational change in the intracellular domain of RPTP alpha led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling.     

PKC regulates the delta2 glutamate receptor interaction with S-SCAM/MAGI-2 protein

Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that delta2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between delta2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.     

Specific binding of coupling factor 1 lacking the delta and epsilon subunits to thylakoids

An improved procedure for the preparation of chloroplast coupling factor 1 (CF1) lacking the delta subunit is described. In addition, CF1 deficient in the epsilon subunit was isolated by a new method and CF1 lacking both of the smaller subunits was prepared. The ability of the subunit-deficient forms and of CF1, either heated or incubated with dithiothreitol to activate its ATPase activity, to bind to thylakoids from which CF1 had been removed was studied. All CF1 preparations bound in a cation-dependent manner to similar extents. CF1 lacking the delta subunit required higher cation concentrations for maximal binding. All preparations competed similarly with control CF1 for binding sites on the depleted membranes. The alpha subunit of all forms of CF1 in solution was rapidly cleaved by trypsin. After reconstitution, however, the alpha subunit of CF1, as well as of the subunit-deficient and the activated forms, was resistant to attack by trypsin. Moreover, treatment of the membranes with either trypsin or N,N'-dicyclohexylcarbodiimide inhibited the binding of all CF1 forms. These results suggest that the binding of the subunit-deficient and activated forms of CF1 is specific. CF1 lacking the epsilon subunit restored neither proton uptake nor ATP synthesis to the depleted membranes. In contrast to our previous results, CF1 lacking the delta subunit was partially effective. Previously, we used a suboptimal Mg2+ concentration for binding the delta-deficient enzyme which we show here was partially deficient in the epsilon subunit. These results show that the delta and epsilon subunits are not required for binding CF1 to the membranes and that the delta subunit is not an absolute requirement for ATP synthesis.     

Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.     

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.     

Bestrophin interacts physically and functionally with protein phosphatase 2A

Bestrophin is a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial cells (RPE). It is encoded by the VMD2 gene, which is mutated in Best macular dystrophy, a disease characterized by a depressed light peak in the electrooculogram. Recently it was proposed that bestrophin is a chloride channel responsible for generating the light peak. To investigate its function further, we immunoaffinity purified a bestrophin complex from RPE lysates and identified bestrophin and the beta-catalytic subunit of protein phosphatase 2A (PP2A) as members of the complex by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Protein-protein interaction between bestrophin and PP2Ac and the structural subunit of PP2A, PR65, was confirmed by reciprocal immunoprecipitation. The C-terminal cytoplasmic domain of bestrophin was sufficient for the interaction with PP2A as demonstrated by a pulldown assay using a fusion of this domain with glutathione S-transferase. Bestrophin was phosphorylated when expressed in RPE-J cells and this phosphorylation was sensitive to okadaic acid. Purified PP2A effectively dephosphorylated bestrophin in vitro. These data suggest that bestrophin is in the signal transduction pathway that modulates the light peak of the electrooculogram, that it is regulated by phosphorylation, and that phosphorylation of bestrophin is in turn regulated by PP2A.