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.     

Interaction of farnesylated PRL-2, a protein-tyrosine phosphatase, with the beta-subunit of geranylgeranyltransferase II.

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the beta-subunit of Rab geranylgeranyltransferase II (betaGGT II). The specific interaction of betaGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with betaGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for betaGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for betaGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the alpha-subunit (alpha) of GGT II, betaGGT II, and PRL-2 resulted in alpha/betaGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous alpha/betaGGT II activity in HeLa cells. Together, these results indicate that the binding of alphaGGT II and PRL-2 to betaGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.     

Trimeric structure of PRL-1 phosphatase reveals an active enzyme conformation and regulation mechanisms

The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. Here, we report the crystal structure of human PRL-1 determined at 2.7A resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes.     

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.     

Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator

The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.     

Association of Arabidopsis type-II ROPs with the plasma membrane requires a conserved C-terminal sequence motif and a proximal polybasic domain

Plant ROPs (or RACs) are soluble Ras-related small GTPases that are attached to cell membranes by virtue of the post-translational lipid modifications of prenylation and S-acylation. ROPs (RACs) are subdivided into two major subgroups called type-I and type-II. Whereas type-I ROPs terminate with a conserved CaaL box and undergo prenylation, type-II ROPs undergo S-acylation on two or three C-terminal cysteines. In the present work we determined the sequence requirement for association of Arabidopsis type-II ROPs with the plasma membrane. We identified a conserved sequence motif, designated the GC-CG box, in which the modified cysteines are flanked by glycines. The GC-CG box cysteines are separated by five to six mostly non-polar residues. Deletion of this sequence or the introduction of mutations that change its nature disrupted the association of ROPs with the membrane. Mutations that changed the GC-CG box glycines to alanines also interfered with membrane association. Deletion of a polybasic domain proximal to the GC-CG box disrupted the plasma membrane association of AtROP10. A green fluorescent protein fusion protein containing the C-terminal 25 residues of AtROP10, including its polybasic domain and GC-CG box, was primarily associated with the plasma membrane but a similar fusion protein lacking the polybasic domain was exclusively localized in the soluble fraction. These data provide evidence for the minimal sequence required for plasma membrane association of type-II ROPs in Arabidopsis and other plant species.     

Upstream polybasic region in peptides enhances dual specificity for prenylation by both farnesyltransferase and geranylgeranyltransferase type I

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of a farnesyl or geranylgeranyl lipid, respectively, near the C-terminus of their protein substrates. FTase and GGTase I differ in both their substrate specificity and magnesium dependence, where the activity of FTase, but not GGTase I, is activated by magnesium. Many protein substrates of these enzymes contain an upstream polybasic region that is proposed to increase the affinity of the substrate and aid in plasma membrane association. Here, we demonstrate that the addition of an upstream polybasic region to a peptide substrate enhances the binding affinity of FTase approximately 4-fold for the peptide but diminishes the catalytic efficiency of the reaction, reflected by decreases in both the prenylation rate constant and kcat/KM. Specifically, the prenylation rate constant decreases 7-fold at 5 mM MgCl2 for the peptide KKKSKTKCVIM (C-terminal sequence of K-Ras4B) in comparison to TKCVIM. This decrease is accompanied by an alteration in the dependence on magnesium, as the K(Mg) increases from 2.2 +/- 0.1 mM for TKCVIM to 11.5 +/- 0.1 mM for KKKSKTKCVIM. The presence of an upstream polybasic region does not significantly affect GGTase I-catalyzed reactions, as only minimal changes are seen in Kd, kcat/KM, and k(chem) values. Thus, the presence of an upstream polybasic region enhances the dual prenylation of these substrates, by decreasing the catalytic efficiency of farnesylation catalyzed by FTase to a level comparable to that of geranylgeranylation catalyzed by GGTase I.     

Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane

Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.     

Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome

PRL-1, -2, and -3 represent a novel class of protein-tyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL-1 and -3 accumulated in a collapsed compact structure around the microtubule-organizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole-like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation.     

Thioredoxin-related Protein 32 (TRP32) Specifically Reduces Oxidized Phosphatase of Regenerating Liver (PRL)

PRL family constitutes a unique class of phosphatases associated with metastasis. The phosphatase activity of PRL has been reported to be important for promoting metastasis, and it is inactivated by reversible oxidation of its catalytic cysteine. Here, we show that TRP32 specifically reduces PRL. Reduction of oxidized PRL in cells is inhibited by 2,4-dinitro-1-chlorobenzene, an inhibitor of TRX reductase. In vitro assays for the reduction of PRL show that only TRP32 can potently reduce oxidized PRL, whereas other TRX-related proteins linked to TRX reductase show little or no reducing activity. Indeed, TRP32 knockdown significantly prolongs the H₂O₂-induced oxidation of PRL. Binding analyses reveal that the unique C-terminal domain of TRP32 is required and sufficient for its direct interaction with PRL. These results suggest that TRP32 maintains the reduced state of PRL and thus regulates the biological function of PRL.     

The double lives of phosphatases of regenerating liver: A structural view of their catalytic and noncatalytic activities

Phosphatases of regenerating liver (PRLs) are protein phosphatases involved in the control of cell growth and migration. They are known to promote cancer metastasis but, despite over 20 years of study, there is still no consensus about their mechanism of action. Recent work has revealed that PRLs lead double lives, acting both as catalytically active enzymes and as pseudophosphatases. The three known PRLs belong to the large family of cysteine phosphatases that form a phosphocysteine intermediate during catalysis. Uniquely to PRLs, this intermediate is stable, with a lifetime measured in hours. As a consequence, PRLs have very little phosphatase activity. Independently, PRLs also act as pseudophosphatases by binding CNNM membrane proteins to regulate magnesium homeostasis. In this function, an aspartic acid from CNNM inserts into the phosphatase catalytic site of PRLs, mimicking a substrate–enzyme interaction. The delineation of PRL pseudophosphatase and phosphatase activities in vivo was impossible until the recent identification of PRL mutants defective in one activity or the other. These mutants showed that CNNM binding was sufficient for PRL oncogenicity in one model of metastasis, but left unresolved its role in other contexts. As the presence of phosphocysteine prevents CNNM binding and CNNM-binding blocks catalytic activity, these two activities are inherently linked. Additional studies are needed to untangle the intertwined catalytic and noncatalytic functions of PRLs. Here, we review the current understanding of the structure and biophysical properties of PRL phosphatases.     

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.     

Low molecular weight protein tyrosine phosphatase and caveolin-1: interaction and isoenzyme-dependent regulation

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are small enzymes that are ubiquitous in many organisms. They are important in biological processes such as cell proliferation, adhesion, migration, and invasiveness. LMW-PTP is expressed in mammalian cells as two isoforms (IF1 and IF2) originating through alternative splicing. We have previously shown that IF2 targets lipid rafts called caveolae and interacts with caveolin-1, their major structural protein. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that have been implicated in a variety of cellular functions, including signal transduction events. Caveolin-1 contains a scaffolding region that contributes to the binding of the protein to the plasma membrane and mediates protein omo- and etero-oligomerization. Interaction of many signaling molecules with the scaffolding domain sequesters them into caveolae and inhibits or suppresses their activities. Caveolin-interacting proteins usually have a typical sequence motif, also present in all the LMW-PTPs, which is characterized by aromatic or large hydrophobic residues in specific positions. We have examined here the interaction of the LMW-PTP isoforms with caveolin-1 and its molecular mechanism, together with the consequences for their tyrosine phosphatase activities. We found that IF1 and IF2 are both capable of interacting with defined regions of caveolin-1 and that their putative caveolin binding sequence motif is not responsible for the association. The formation of LMW-PTP/caveolin-1 complexes is accompanied by modulation of the enzyme activities, and the inhibitory effect elicited against IF1 is stronger than that against IF2. The caveolin scaffolding domain is directly involved in the observed phenomena.     

苎麻小分子G蛋白Racl基因的克隆及表达分析

植物Rac是植物中特有的小分子G蛋白,我们从苎麻转录组中获得一个小分子G蛋白基因eDNA,．5部分序列,设计引物后采用RT-PCR结合RACE技术克隆了该基因的cDNA。序列分析表明,所克隆的RaclcDNA全长为l043bp,包括594bp开放阅读框、214bp的3’端非编码区和235bp的5’端非编码区,能编码一个197氨基酸的推导蛋白。该蛋白包含G蛋白典型的效应因子结合位点、GTP／GDP结合位点和碱性氨基酸区,c末端具有保守的异戊烯基化位,CSIL。采用半定量RT-PCR分析了该基因在5个苎麻品种及不同组织器官中的表达情况,结果表明Racl基因在苎麻根、茎、叶中均有表达,其中在叶中的表达量最高。纤维木质素含量不同的品种中,Racl基因的表达量存在明显差异。木质素含量高的品种具有较高的Racl基因表达,表明该基因可能在苎麻木质素合成过程中发挥作用。     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.     

A Conserved Motif Mediates both Multimer Formation and Allosteric Activation of Phosphoglycerate Mutase 5

Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5.