The conserved immunoglobulin domain controls the subcellular localization of the homophilic adhesion receptor protein-tyrosine phosphatase mu

The receptor protein-tyrosine phosphatase mu (PTPmu) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPmu resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPmu, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPmu is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPmu to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPmu in subconfluent endothelial cells when homophilic binding to another PTPmu molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPmu and the importance of such control for the maintenance of normal cell physiology.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

A novel binding pocket in the D2 domain of protein tyrosine phosphatase mu (PTPmu) guides AI screen to identify small molecules that modulate tumour cell adhesion,growth and migration

Approximately 40% of people will get cancer in their lifetime in the US, and 20% are predicted to die from the condition when it is invasive and metastatic. Targeted screening for drugs that interact with proteins that drive cancer cell growth and migration can lead to new therapies. We screened molecular libraries with the AtomNet® AI-based drug design tool to identify compounds predicted to interact with the cytoplasmic domain of protein tyrosine phosphatase mu. Protein tyrosine phosphatase mu (PTPmu) is proteolytically downregulated in cancers such as glioblastoma generating fragments that stimulate cell survival and migration. Aberrant nuclear localization of PTPmu intracellular fragments drives cancer progression, so we targeted a predicted drug-binding site between the two cytoplasmic phosphatase domains we termed a D2 binding pocket. The function of the D2 domain is controversial with various proposed regulatory functions, making the D2 domain an attractive target for the development of allosteric drugs. Seventy-five of the best-scoring and chemically diverse computational hits predicted to interact with the D2 binding pocket were screened for effects on tumour cell motility and growth in 3D culture as well as in a direct assay for PTPmu-dependent adhesion. We identified two high-priority hits that inhibited the migration and glioma cell sphere formation of multiple glioma tumour cell lines as well as aggregation. We also identified one activator of PTPmu-dependent aggregation, which was able to stimulate cell migration. We propose that the PTPmu D2 binding pocket represents a novel regulatory site and that inhibitors targeting this region may have therapeutic potential for treating cancer.     

Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling

Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.     

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.     

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.     

A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta (PTPzeta) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.     

The pleckstrin homology (PH) domain of the Arf exchange factor Brag2 is an allosteric binding site

Brag2, a Sec7 domain (sec7d)-containing guanine nucleotide exchange factor, regulates cell adhesion and tumor cell invasion. Brag2 catalyzes nucleotide exchange, converting Arf·GDP to Arf·GTP. Brag2 contains a pleckstrin homology (PH) domain, and its nucleotide exchange activity is stimulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we determined kinetic parameters for Brag2 and examined the basis for regulation by phosphoinositides. Using myristoylated Arf1·GDP as a substrate, the k(cat) was 1.8 ± 0.1/s as determined by single turnover kinetics, and the K(m) was 0.20 ± 0.07 μm as determined by substrate saturation kinetics. PIP(2) decreased the K(m) and increased the k(cat) of the reaction. The effect of PIP(2) required the PH domain of Brag2 and the N terminus of Arf and was largely independent of Arf myristoylation. Structural analysis indicated that the linker between the sec7d and the PH domain in Brag2 may directly contact Arf. In support, we found that a Brag2 fragment containing the sec7d and the linker was more active than sec7d alone. We conclude that Brag2 is allosterically regulated by PIP(2) binding to the PH domain and that activity depends on the interdomain linker. Thus, the PH domain and the interdomain linker of Brag2 may be targets for selectively regulating the activity of Brag2.     

The human protein-tyrosine phosphatase PTP alpha/LRP gene (PTPA) is assigned to chromosome 20p13.

In situ hybridization was employed to localize a cDNA probe from the human protein-tyrosine phosphatase PTP alpha/LRP to human metaphase chromosomes. The PTPA gene has been localized to chromosome 20p13.     

Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.

A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S. cerevisiae YVH1 gene. The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas. The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function. The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain. Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice.

The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.     

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.     

The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gbeta gamma binding site

G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.     

The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site

Hsp90 is a ubiquitous, well-conserved molecular chaperone involved in the folding and stabilization of diverse proteins. Beyond its capacity for general protein folding, Hsp90 influences a wide array of cellular signaling pathways that underlie key biological and disease processes. It has been proposed that Hsp90 functions as a molecular clamp, dimerizing through its carboxy-terminal domain and utilizing ATP binding and hydrolysis to drive large conformational changes including transient dimerization of the amino-terminal and middle domains. We have determined the 2.6 A X-ray crystal structure of the carboxy-terminal domain of htpG, the Escherichia coli Hsp90. This structure reveals a novel fold and that dimerization is dependent upon the formation of a four-helix bundle. Remarkably, proximal to the helical dimerization motif, each monomer projects a short helix into solvent. The location, flexibility, and amphipathic character of this helix suggests that it may play a role in substrate binding and hence chaperone activity.     

The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site).

Glutathione S -transferases (GSTs) play a pivotal role in the detoxification of foreign chemicals and toxic metabolites. They were originally termed ligandins because of their ability to bind large molecules (molecular masses >400 Da), possibly for storage and transport roles. The location of the ligandin site in mammalian GSTs is still uncertain despite numerous studies in recent years. Here we show by X-ray crystallography that the ligandin binding site in human pi class GST P1-1 occupies part of one of the substrate binding sites. This work has been extended to the determination of a number of enzyme complex crystal structures which show that very large ligands are readily accommodated into this substrate binding site and in all, but one case, causes no significant movement of protein side-chains. Some of these molecules make use of a hitherto undescribed binding site located in a surface pocket of the enzyme. This site is conserved in most, but not all, classes of GSTs suggesting it may play an important functional role.     

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.     

The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium.

The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding p6 mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition.