Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion

The PRL (phosphatase of regenerating liver) phosphatases constitute a novel class of small, prenylated phosphatases that are implicated in promoting cell growth, differentiation, and tumor invasion, and represent attractive targets for anticancer therapy. Here we describe the crystal structures of native PRL-1 as well as the catalytically inactive mutant PRL-1/C104S in complex with sulfate. PRL-1 exists as a trimer in the crystalline state, burying 1140 A2 of accessible surface area at each dimer interface. Trimerization creates a large, bipartite membrane-binding surface in which the exposed C-terminal basic residues could cooperate with the adjacent prenylation group to anchor PRL-1 on the acidic inner membrane. Structural and kinetic analyses place PRL-1 in the family of dual specificity phopsphatases with closest structural similarity to the Cdc14 phosphatase and provide a molecular basis for catalytic activation of the PRL phosphatases. Finally, native PRL-1 is crystallized in an oxidized form in which a disulfide is formed between the active site Cys104 and a neighboring residue Cys49, which blocks both substrate binding and catalysis. Biochemical studies in solution and in the cell support a potential regulatory role of this intramolecular disulfide bond formation in response to reactive oxygen species such as H2O2.     

Structural insights into molecular function of the metastasis-associated phosphatase PRL-3

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity.     

Mycobacterium tuberculosis protein tyrosine phosphatase PtpB structure reveals a diverged fold and a buried active site

Intracellular pathogenic bacteria manipulate host signal transduction pathways to facilitate infection. Mycobacterium tuberculosis protein tyrosine phosphatases (PTPs) PtpA and PtpB are thought to be secreted into host cells and interfere with unidentified signals. To illuminate the mechanisms of regulation and substrate recognition, we determined the 1.7 A resolution crystal structure of PtpB in complex with the product phosphate. The protein adopts a simplified PTP fold, which combines features of the conventional PTPs and dual-specificity phosphatases. PtpB shows two unusual elaborations--a disordered, acidic loop and a flexible, two-helix lid that covers the active site--that are specific to mycobacterial orthologs. Biochemical studies suggest that substrate mimicry in the lid may protect the phosphatase from oxidative inactivation. The insertion and deletion of large structural elements in PtpB suggest that, outside the active site module, the PTP family is under unusual selective pressure that promotes changes in overall structure.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Phosphatase activity, trimerization, and the C-terminal polybasic region are all required for PRL1-mediated cell growth and migration

The phosphatase of regenerating liver (PRL) phosphatases are implicated in a number of tumorigenesis and metastasis processes. The PRLs are unique among protein-tyrosine phosphatases in that they have extremely low phosphatase activity, a high propensity for trimer formation, and a polybasic region that precedes the C-terminal prenylation motif. To investigate the functional significance of these distinctive biochemical and structural features, we established a cell-based system in which ectopic PRL1 expression increased cell proliferation and migration, whereas knockdown of endogenous PRL1 abrogated these cellular activities. We showed that the intrinsic PRL1 phosphatase activity is obligatory for its biological function. We provided evidence that trimerization may be a general property for all PRL enzymes, and that PRL1 trimer formation is essential for the PRL1-mediated cell growth and migration. This finding indicates a novel mechanism for phosphatase regulation. We further demonstrated that the conserved C-terminal polybasic region is important for specific phosphoinositide recognition by PRL1. Both the polybasic residues and the adjacent prenylation motif are required for proper PRL1 subcellular localization and full biological activity.     

Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction

Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of active-site cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.     

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.     

Exploring the cause of the inhibitor 4AX attaching to binding site disrupting protein tyrosine phosphatase 4A1 trimerization by molecular dynamic simulation

Abstract

Ectopic overexpression of protein tyrosine phosphatase of liver regeneration-1 (PTP4A1, also called PRL-1) markedly enhanced hepatocellular carcinoma (HCC) cells migration and invasion. The PTP4A1 trimerization played a vital role in mediating cell proliferation and motility. Biochemical and structural studies have proved that the compound 4AX, a well-known inhibitor for PRL1, directly binds to the PTP4A1 trimer interface and obstructs trimer formation of PTP4A1. However, the molecular basis of the ligand-4AX inhibition on PTP4A1 trimer conformations remains unclear. In this study, the docking analysis and the molecular dynamics simulation (MD simulation) study were performed to investigate how the molecule binding at each interface disrupted the trimer formation. The results suggested that the ligand-4AX attaching to the binding site changed the conformation of A:Q131, A:Q135 in the AC interface, C:R18, C:P96 in the CA interface and B:Q131 in the BA interface, leading to the weak interactions between subunits and thus resulting in the disruption of the PTP4A1 trimerization.     

PRL-3 phosphatase and cancer metastasis

The deregulated expression of members of the phosphatase of regenerating liver (PRL) family has been implicated in the metastatic progression of multiple human cancers. Importantly, PRL-1 and PRL-3 both possess the capacity to drive key steps in metastatic progression. Yet, little is known about the regulation and oncogenic mechanisms of this emerging class of dual-specificity phosphatases. This prospect article details the involvement of PRLs in the metastatic cascade, the regulatory mechanisms controlling PRL expression, and recent efforts in the characterization of PRL-modulated pathways and substrates using biochemical and high-throughput approaches. Current advances and future prospects in anti-cancer therapy targeting this family are also discussed.     

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.     

Crystal structure of the low-humidity form of aspartame sweetener.

The low-humidity IB crystal form of aspartame (L-alphaaspartyl-L-phenylalanine methyl ester) is prepared via humidity-induced transition from the highly hydrated IA crystal form and is used widely as a sweetener. The crystal structure of the low-humidity IB form is determined at 1.05 A resolution (0.476 A(-1) in maximum sintheta/lambda) from an extremely fine fibrous crystal using synchrotron radiation. There are three aspartame molecules and two water molecules in the asymmetric unit of the monoclinic space group P2(1). Each aspartame molecule adopts an almost identical extended conformation which is commonly observed in other crystal forms of aspartame. Three aspartame molecules are assembled into a triangular trimer, and trimer units are stacked along the b-axis via hydrogen-bonding and electrostatic interactions in the main chains and also via hydrophobic contacts in the phenyl side-chains. Six trimer units are related by pseudo 6(1)-screw axis symmetry and form a hydrophilic channel at their center. The hydrophilic channel in the IB form contains only four water molecules in the unit cell, compared with 16 in the IA form. Although the IB form exhibits a trimer structure similar to that of the IA form, one aspartame molecule is rotated by approximately equals 20 degrees from the orientation in the IA form. This arrangement of the molecule implies that the humidity-induced transition is accompanied by a flapping motion of its methyl ester group. These structural differences may imply the stepwise transition from the IA to the IB forms.     

Visualizing active-site dynamics in single crystals of HePTP: opening of the WPD loop involves coordinated movement of the E loop

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an ‘atypically open’ conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.     

PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth.

PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.     

Protein tyrosine phosphatases: structure-function relationships

Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.     

The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis

PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics.     

Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose

The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively. A4-beta-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.     

Crystal structure of a mammalian purple acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is a mammalian di-iron- containing enzyme that belongs to the family of purple acid phosphatases (PAP). It is highly expressed in a limited number of tissues, predominantly in bone-resorbing osteoclasts and in macrophages of spleen. We have determined the crystal structure of rat TRAP in complex with a phosphate ion to 2.7 A resolution. The fold resembles that of the catalytic domain of kidney bean purple acid phosphatase (KBPAP), although the sequence similarity is limited to the active site residues. A surface loop near the active site is absent due to proteolysis, leaving the active-site easily accessible from the surrounding solvent. This, we believe, gives a structural explanation for the observed proteolytic activation of TRAP. The current structure was determined at a relatively high pH and without any external reducing agents. It is likely that it represents an oxidized and therefore catalytically inactive form of the enzyme.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.