Protein tyrosine phosphatases (PTPases) and kinases coregulate the critical levels of phosphorylation necessary for intracellular signalling, cell growth and differentiation. Yersinia, the causative bacteria of the bubonic plague and other enteric diseases, secrete an active PTPase, Yop51, that enters and suppresses host immune cells. Though the catalytic domain is only approximately 20% identical to human PTP1B, the Yersinia PTPase contains all of the invariant residues present in eukaryotic PTPases, including the nucleophilic Cys 403 which forms a phosphocysteine intermediate during catalysis. We present here structures of the unliganded (2.5 A resolution) and tungstate-bound (2.6 A) crystal forms which reveal that Cys 403 is positioned at the centre of a distinctive phosphate-binding loop. This loop is at the hub of several hydrogen-bond arrays that not only stabilize a bound oxyanion, but may activate Cys 403 as a reactive thiolate. Binding of tungstate triggers a conformational change that traps the oxyanion and swings Asp 356, an important catalytic residue, by approximately 6 A into the active site. The same anion-binding loop in PTPases is also found in the enzyme rhodanese.

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR,68..,,, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR, 9ss. (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affmity binding and catalysis. The K. value for EGFR,85_.,, decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.The tyrosine phosphorylation "status" of a cell is maintained by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) (1, 2). The first PTPase to be purified and sequenced was a 35-kDa protein (PTP 1B) from human placenta (3, 4). PTP1B was shown to share amino acid sequence homology with the cytoplasmic domain of the leukocyte cell surface glycoprotein CD45 (5). CD45 was subsequently shown to have tyrosine phosphatase activity (6). This discovery drew attention to the possible biological role of the PTPases in controlling signal transduction within the cell. Isolation and characterization of cDNA by low stringency hybridization and polymerase chain reaction demonstrated that PTPases constitute a large diversified family of catalysts that can be divided into two structurally distinct groups. One group generally has an extracellular domain, transmembrane spanning region, as well as two duplicated cytoplasmic PTPase domains. The other group of PTPases corresponds to the intracellular family of enzymes that have a single PTPase domain (2).Many PTPases have been cloned but limited information is available about their functions, mechanisms of catalysis, or substrate specificities. One of the central questions in protein phosphorylation is how kinases and phosphatases distinguish the diversity of substrates that they encounter in the cell. In the case of protein kinases, the amino acid sequence surrounding the phosphorylation site plays a crucial role in determining substrate specificity (7,8). For example, the cAMP-dependent protein kinase has a strong preference for phosphorylation of Ser residues that are located two or three residues to the COOH-terminal side of basic amino acids (most commonly Arg). Tyr...

In this report, we demonstrated that the Yersinia protein tyrosine phosphatase (PTPase) could be inactivated by the alkylating agent iodoacetate. The enzyme modification was selective, and the covalent attachment was stoichiometric. The residue that was labeled by iodoacetate was shown to be Cys403, which was the same catalytically essential residue identified by site-directed mutagenesis [Guan, K. L., & Dixon, J.E. (1990) Science 249, 553-556]. The rate of iodoacetate modification decreased as the ionic strength of the media increased. There was no significant D2O solvent isotope effect associated with the inactivation of the enzyme, suggesting that thiol anion of Cys403 reacted as a nucleophile. The Yersinia PTPase also displayed differential reactivity (940-fold) toward iodoacetate over iodoacetamide. This indicates that residues within the active site of the enzyme are positively charged. The pKa of the active site thiol group was determined to be 4.67. The low pKa value suggests that ionic interactions are important in stabilizing the thiolate anion. One candidate residue for this stabilization is the invariant histidine (His402) found in all PTPases. Substitutions of His402 with Asn or Ala altered the active site thiol pKa to 5.99 and 7.35, respectively. Interestingly, the active site thiol in the mutants also showed enhanced reactivity toward iodoacetate. The second-order rate constants for the inactivation of the wild-type enzyme, H402N, and H402A were 59.7, 3305, and 1763 M-1 min-1, respectively.

The structural requirements of substrates for two recombinant protein tyrosine phosphatases (PTPases) are probed using various-sized synthetic phosphotyrosine (pY)-containing peptides corresponding to the autophosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due to its favorable kinetic constants. The contribution of individual amino acids on both sides of pY to binding and catalysis was assessed by kinetic analysis using a continuous, spectrophotometric assay. For both Yersinia PTPase and a soluble recombinant mammalian PTPase of 323 amino acid residues (rat PTP1), efficient binding and catalysis required six amino acids including the pY residue, i.e., four residues N-terminal to pY and one residue C-terminal to pY. Thus, PTPase substrate specificity is primarily dictated by residues to the N-terminal side of pY. The pY moiety and the rest of the peptide interact with PTPases in a cooperative manner. The presence of pY in the peptide substrate is necessary but not sufficient for high-affinity binding, since phosphotyrosine and other simple aryl phosphates exhibit weak binding, and dephosphorylated peptides do not bind to PTPases. Two variations on the pY moiety are also examined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the phosphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, whereas the phosphonomethylphenylalanine analog in which the tyrosyl oxygen is replaced by a CH2 group is a competitive and nonhydrolyzable inhibitor, with Ki values of 18.6 and 10.2 microM, respectively, for the Yersinia PTPase and the rat PTP1.

The Yersinia protein tyrosine phosphatase (PTPase) was identified in the genus of bacteria responsible for the plague or the Black Death and was shown to be essential for pathogenesis. The three-dimensional structure of the catalytic domain of the Yersinia PTPase has been solved, and this information along with a detailed kinetic analysis has led to a better understanding of the catalytic mechanism of the PTPase. Mutational and chemical modification experiments have established that an invariant Cys residue (Cys403) is directly involved in formation of a covalent phosphoenzyme intermediate. We have shown that Arg409 plays a critical role in PTPase action and that the Cys(X)5Arg active site motif forms a phosphate-binding loop which appears to represent the essential features necessary for catalysis by the PTPases, the dual specific phosphatases, and the low molecular weight acid phosphatases.

The protein-tyrosine phosphatases (PTPases) superfamily consists of tyrosine-specific phosphatases, dual specificity phosphatases, and the low-molecular-weight phosphatases. They are modulators of signal transduction pathways that regulate numerous cell functions. Malfunction of PTPases have been linked to a number of oncogenic and metabolic disease states, and PTPases are also employed by microbes and viruses for pathogenicity. There is little sequence similarity among the three subfamilies of phosphatases. Yet, three-dimensional structural data show that they share similar conserved structural elements, namely, the phosphate-binding loop encompassing the PTPase signature motif (H/V)C(X)5R(S/T) and an essential general acid/base Asp residue on a surface loop. Biochemical experiments demonstrate that phosphatases in the PTPase superfamily utilize a common mechanism for catalysis going through a covalent thiophosphate intermediate that involves the nucleophilic Cys residue in the PTPase signature motif. The transition states for phosphoenzyme intermediate formation and hydrolysis are dissociative in nature and are similar to those of the solution phosphate monoester reactions. One strategy used by these phosphatases for transition state stabilization is to neutralize the developing negative charge in the leaving group. A conformational change that is restricted to the movement of a flexible loop occurs during the catalytic cycle of the PTPases. However, the relationship between loop dynamics and enzyme catalysis remains to be established. The nature and identity of the rate-limiting step in the PTPase catalyzed reaction requires further investigation and may be dependent on the specific experimental conditions such as temperature, pH, buffer, and substrate used. In-depth kinetic and structural analysis of a representative number of phosphatases from each group of the PTPase superfamily will most likely continue to yield insightful mechanistic information that may be applicable to the rest of the family members.

Cas is a member of the focal adhesion complex. Phosphorylation of Cas by Src is an important event leading to cell transformation. Using mass spectrometry, we have mapped 11 sites in Cas that are phosphorylated by Src. These sites are all located between residues 132 and 414 of Cas, in a region that is required for binding to a number of other proteins including Crk. We tested synthetic peptides modeled on Cas phosphorylation sites, and found that the sequence containing tyrosine 253 was phosphorylated by Src most efficiently. Using cells derived from Cas-deficient mice, we confirmed that Cas greatly enhanced the ability of Src to transform cells. Phosphorylation of Cas on tyrosine 253 was not required for Src to increase growth rate, suppress contact inhibition, or suppress anchorage dependence. Yet, in contrast to these growth characteristics, phosphorylation of Cas on tyrosine 253 was required for Src to promote cell migration. Thus, a single phosphorylation site on this focal adhesion adaptor protein can effectively separate cell migration from other transformed growth characteristics.Normal cells have intrinsic controls that limit their movement and growth, such as restraints imposed by cell cycle checkpoints. Cells must receive cues from their surrounding environment to override these restraints. Thus, cell growth normally relies on signals mediated from the extracellular matrix through integrins (1-3).In contrast to normal cells, tumor cells ignore their surrounding environment, which allows them to overcome contact growth inhibition, lose anchorage dependence, and migrate to foreign tissues and organs. The degree to which tumor cells grow and migrate correlates with their aggressive potential (1, 2). Therefore, it is important to understand how extracellular signals guide cell behavior and how transformation ablates the need for integrin signaling.Integrins transmit signals from the extracellular matrix to inside of the cell. Integrin signaling relies on a complex of associated kinases including focal adhesion kinase (FAK) 1 and Src and adaptor proteins including Grb2, Shc, paxillin, and Cas (4, 5). Cas is an important component of the integrin signaling network (6). Cas was originally identified as a protein phosphorylated in v-src-transformed cells (7).Cas has several structural motifs including an SH3 domain, proline-rich regions, and a cluster of tyrosine phosphorylation consensus sites that act as SH2 binding motifs (6,8). FAK binds to the SH3 domain in the amino-terminal part of Cas (9 -11), whereas Src binds to a proline-rich region and a phosphorylated tyrosine residue at the carboxyl end of Cas (12, 13). Although FAK may phosphorylate Cas in certain cases (14, 15), the biological activity of Cas depends on its phosphorylation by Src (16 -18). After phosphorylation, Cas associates with a number of proteins, including Crk, Src, phosphatidylinositol 3-kinase, Nck, and phospholipase C␥, via SH2 binding motifs (8,10,19).Phosphorylation of Cas by Src plays a critical role in cell transformation (20,...

Two continuous assays for protein tyrosine phosphatases (PTPases) have been developed using phosphotyrosine containing peptide substrates. These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at 282 nm or the fluorescence at 305 nm of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation. The procedure is convenient and efficient, since both kcat and Km values can be obtained in a single run. The difference absorption coefficient (delta epsilon) at 282 nm is relatively insensitive to the pH of the reaction media. These techniques were applied to two homogeneous recombinant PTPases employing six phosphotyrosine-containing peptides. Km and kcat values obtained from the progress curve analysis were similar to those determined by the traditional initial rate inorganic phosphate assay. The peptides corresponding to autophosphorylation sites in Neu, p56lck, and p60src proteins show distinct behavior with the Yersinia PTPase, Yop51*, and the mammalian PTPase (PTP1U323). In both cases, the kcat values were relatively constant for all the peptides tested whereas the Km values were very sensitive to the amino acid sequence surrounding the tyrosine residue, especially in the case of Yop51*. Thus, both Yop51* and PTP1U323 show differential recognition of the phosphotyrosyl residues in the context of distinct primary structure of peptide substrates.