ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.     

The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.     

Dimerization of the scaffolding protein ZO-1 through the second PDZ domain

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH(2)-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the beta-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3.     

Solution structure of the PDZ2 domain from cytosolic human phosphatase hPTP1E complexed with a peptide reveals contribution of the beta2-beta3 loop to PDZ domain-ligand interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.     

Structural insights into the PIP2 recognition by syntenin-1 PDZ domain

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP₂) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the α2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP₂. Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP₂ with the PDZ domain of the well-known PIP₂-binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP₂ could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP₂-mediated PDZ ligand-binding regulation.     

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.     

Sequence-specific 1H, 13C, and 15N assignment of the extended PDZ3 domain of the protein tyrosine phosphatase basophil-like PTP-BL

Protein tyrosine phosphatase basophil-like (PTP-BL), also known as PTPN13, represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the complete resonance assignments of the extended PDZ3 domain of PTP-BL. These assignments provide a basis for the detailed structural investigation of the interaction between the PDZ domains of PTP-BL as well as of their interaction with ligands. It will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTB-BL.     

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.     

Novel mode of ligand recognition by the Erbin PDZ domain

Erbin contains a class I PDZ domain that binds to the C-terminal region of the receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the human Erbin PDZ bound to the peptide EYLGLDVPV corresponding to the C-terminal residues 1247-1255 of human ErbB2 has been determined at 1.25-A resolution. The Erbin PDZ deviates from the canonical PDZ fold in that it contains a single alpha-helix. The isopropyl group of valine at position -2 of the ErbB2 peptide interacts with the Erbin Val(1351) and displaces the peptide backbone away from the alpha-helix, elucidating the molecular basis of class II ligand recognition by a class I PDZ domain. Strikingly, the phenolic ring of tyrosine -7 enters into a pocket formed by the extended beta 2-beta 3 loop of the Erbin PDZ. Phosphorylation of tyrosine -7 abolishes this interaction but does not affect the binding of the four C-terminal peptidic residues to PDZ, as revealed by the crystal structure of the Erbin PDZ complexed with a phosphotyrosine-containing ErbB2 peptide. Since phosphorylation of tyrosine -7 plays a critical role in ErbB2 function, the selective binding and sequestration of this residue in its unphosphorylated state by the Erbin PDZ provides a novel mechanism for regulation of the ErbB2-mediated signaling and oncogenicity.     

Catalytic inactivation of protein tyrosine phosphatase CD45 and protein tyrosine phosphatase 1B by polyaromatic quinones

Polyaromatic quinones, such as the environmental pollutants 9,10-phenanthrenediones, elicit a wide range of responses including growth inhibition, immune suppression, and glucose normalization in diabetic models. Yet the molecular mechanisms behind these effects remain controversial. Here we report that many of them are oxygen-dependent and catalytic inactivators of protein tyrosine phosphatases (PTP). Under aerobic conditions, the PTP inactivation by 2-nitro-9,10-phenanthrenedione followed a pseudo-first-order process, with the rate of inactivation increasing nearly linearly with increasing inhibitor concentration, yielding apparent inactivation rate constants of 4300, 387, and 5200 M(-1) s(-1) at pH 7.2 against CD45, PTP1B, and LAR, respectively. The rate of CD45 inactivation increased approximately 25-fold from pH 6.0 to 7.5, with complete inactivation achieved using a catalytic amount (0.05 molar equiv) of the inhibitor. The quinone-catalyzed CD45 inactivation was prevented by catalase or superoxide dismutase. Inactivated CD45 after (125)I-9,10-phenanthrenedione treatment carried no radioactivity, indicating the absence of a stable inhibitor/enzyme complex. The activity of inactivated CD45 was partially restored ( approximately 10%) by hydroxylamine or dithiothreitol, supporting the presence of a small population of sulfenic acid or sulfenyl-amide species. Treatment of PTP1B with 2-nitro-9,10-phenanthrenedione resulted in the specific and sequential oxidation of the catalytic cysteine to the sulfinic and sulfonic acid. These results suggest that reactive oxygen species and the semiquinone radical, continuously generated during quinone-catalyzed redox cycling, mediate the specific catalytic cysteine oxidation. Naturally occurring quinones may act as efficient regulators of protein tyrosine phosphorylation in biological systems. Aberrant phosphotyrosine homeostasis resulting from continued polyaromatic hydrocarbon quinone exposure may play a significant role in their disease etiology.     

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

Regulation of protein tyrosine phosphatase 1B by sumoylation

Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.     

Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B

Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

ESI-MS and FTIR studies of the interaction between the second PDZ domain of hPTP1E and target peptides.

The specificity of interaction between the second PDZ domain of human protein tyrosine phosphatase1E (PDZ2) and a C-terminal peptide, ENEQVSAV, from the guanine nucleotide exchange factor RA-GEF-2 was investigated using Fourier transform infrared (FTIR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Specificity of the binding interaction and the importance of Ser in the -2 position of the target peptide were demonstrated using alternate peptides ENEQVCAV and KDDEVYYV. FTIR-monitored thermal denaturation in the amide I region showed a 10 degrees C increase in melting temperature (Tm) for the PDZ2-ENEQVSAV complex compared with that of free PDZ2, and the spectra revealed increased absorption in the beta-sheet region (1628 cm(-1)) of PDZ2 on peptide binding. Neither of these results were observed with peptides containing either Cys or Tyr in the -2 position. Complex formation with the Ser-containing peptide was further demonstrated by direct measurement of a 1:1 PDZ-peptide complex by ESI-MS in 100% aqueous solutions without the need for organic co-solvents. Our results demonstrate that even a single atom (O --> S) substitution from Ser to Cys in the -2 position disrupts C-terminal peptide binding to PDZ2.     

Crystal structure of human protein tyrosine phosphatase SHP-1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011. © 2011 Wiley‐Liss, Inc.     

The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL

The small adaptor protein RIL consists of two segments, the C-terminal LIM and the N-terminal PDZ domain, which mediate multiple protein-protein interactions. The RIL LIM domain can interact with PDZ domains in the protein tyrosine phosphatase PTP-BL and with the PDZ domain of RIL itself. Here, we describe and characterise the interaction of the RIL PDZ domain with the zyxin-related protein TRIP6, a protein containing three C-terminal LIM domains. The second LIM domain in TRIP6 is sufficient for a strong interaction with RIL. A weaker interaction with the third LIM domain in TRIP6, including the proper C-terminus, is also evident. TRIP6 also interacts with the second out of five PDZ motifs in PTP-BL. For this interaction to occur both the third LIM domain and the proper C-terminus are necessary. RNA expression analysis revealed overlapping patterns of expression for TRIP6, RIL and PTP-BL, most notably in tissues of epithelial origin. Furthermore, in transfected epithelial cells TRIP6 can be co-precipitated with RIL and PTP-BL PDZ polypeptides, and a co-localisation of TRIP6 and RIL with Factin structures is evident. Taken together, PTP-BL, RIL and TRIP6 may function as components of multi-protein complexes at actin-based sub-cellular structures.     

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.