The crystal structures of a cysteine-215-->serine mutant of protein tyrosine phosphatase 1B complexed with high-affinity peptide substrates corresponding to an autophosphorylation site of the epidermal growth factor receptor were determined. Peptide binding to the protein phosphatase was accompanied by a conformational change of a surface loop that created a phosphotyrosine recognition pocket and induced a catalytically competent form of the enzyme. The phosphotyrosine side chain is buried within the period and anchors the peptide substrate to its binding site. Hydrogen bonds between peptide main-chain atoms and the protein contribute to binding affinity, and specific interactions of acidic residues of the peptide with basic residues on the surface of the enzyme confer sequence specificity.

The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a ''substrate trap.'' Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substratebinding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligandindependent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.The protein tyrosine phosphatases (PTPs) are a structurally diverse family of receptor-like and nontransmembrane enzymes that have been implicated in the control of numerous physiological processes, including growth and differentiation (1, 2). Approximately 75 PTPs have been identified to date. These enzymes are characterized by the presence of a conserved catalytic domain of Ϸ240 residues which contains the unique signature motif,

Protein tyrosine phosphatases (PTPs) constitute a family of receptor-like and cytoplasmic signal transducing enzymes that catalyze the dephosphorylation of phosphotyrosine residues and are characterized by homologous catalytic domains. The crystal structure of a representative member of this family, the 37-kilodalton form (residues 1 to 321) of PTP1B, has been determined at 2.8 A resolution. The enzyme consists of a single domain with the catalytic site located at the base of a shallow cleft. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. This site is formed from an 11-residue sequence motif that is diagnostic of PTPs and the dual specificity phosphatases, and that contains the catalytically essential cysteine and arginine residues. The position of the invariant cysteine residue within the phosphate binding site is consistent with its role as a nucleophile in the catalytic reaction. The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation.

Findings of surgical lung biopsy (SLB) are important in categorizing patients with idiopathic interstitial pneumonia (IIP). We investigated whether histologic variability would be evident in SLB specimens from multiple lobes in patients with IIP. SLBs from 168 patients, 109 of whom had multiple lobes biopsied, were reviewed by three pathologists. A diagnosis was assigned to each lobe. A different diagnosis was found between lobes in 26% of the patients. Patients with usual interstitial pneumonia (UIP) in all lobes were categorized as concordant for UIP (n = 51) and those with UIP in at least one lobe were categorized as discordant for UIP (n = 28). Patients with nonspecific interstitial pneumonia (NSIP) in all lobes were categorized as having fibrotic (n = 25) or cellular NSIP (n = 5). No consistent distribution of lobar histology was noted. Patients concordant for UIP were older (63 +/- 9 [mean +/- SD] yr; p < 0.05 as compared with all other groups) than those discordant for UIP (57 +/- 12 yr) or with fibrotic NSIP (56 +/- 11 yr) or cellular NSIP (50 +/- 9 yr). Semiquantitative high-resolution computed tomography demonstrated a varied profusion of fibrosis (p < 0.05 for all group comparisons), with more fibrosis in concordant UIP (2.13 +/- 0.62) than in discordant UIP (1.42 +/- 0.73), fibrotic NSIP (0.83 +/- 0.58), or cellular NSIP (0.44 +/- 0.42). Survival was better for patients with NSIP than for those in both UIP groups (p < 0.001), although survival in the two UIP groups was comparable (p = 0.16). Lobar histologic variability is frequent in patients with IIP, patients with a histologic pattern of UIP in any lobe should be classified as having UIP.

Idiopathic interstitial pneumonias are a diverse group of lung diseases with varied prognoses. We hypothesized that changes in physiologic and radiographic parameters would predict survival. We retrospectively examined 80 patients with usual interstitial pneumonia and 29 patients with nonspecific interstitial pneumonia. Baseline characteristics were examined together with 6-month change in forced vital capacity, diffusing capacity for carbon monoxide, and ground glass infiltrate and fibrosis on high resolution computed tomography. Patients with usual interstitial pneumonia were more likely to have a statistically significant or marginally significant decline in lung volume, diffusing capacity for carbon monoxide, and an increase in ground glass infiltrates (p < or = 0.08) compared with patients with nonspecific interstitial pneumonia. For patients with usual interstitial pneumonia, change in forced vital capacity was the best physiologic predictor of mortality (p = 0.05). In a multivariate Cox proportional hazards model controlling for histopathologic diagnosis, gender, smoking history, baseline forced vital capacity, and 6-month change in forced vital capacity, a decrease in forced vital capacity remained an independent risk factor for mortality (decrease > 10%; hazard ratio 2.47; 95% confidence interval 1.29, 4.73; p = 0.006). We conclude that a 6-month change in forced vital capacity gives additional prognostic information to baseline features for patients with idiopathic interstitial pneumonia.

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Patients with idiopathic interstitial pneumonias (IIPs) can be subdivided into groups based on the histological appearance of lung tissue obtained by surgical biopsy. The quantitative impact of histological diagnosis, baseline factors and response to therapy on survival has not been evaluated.Surgical lung biopsy specimens from 168 patients with suspected IIP were reviewed according to the latest diagnostic criteria. The impact of baseline clinical, physiological, radiographic and histological features on survival was evaluated using Cox regression analysis. The predictive value of honeycombing on high-resolution computed tomography (HRCT) as a surrogate marker for usual interstitial pneumonia (UIP) was examined. The response to therapy and survival of 39 patients treated prospectively with high-dose prednisone was evaluated.The presence of UIP was the most important factor influencing mortality. The risk ratio of mortality when UIP was present was 28.46 (95% confidence interval (CI) 5.5-148.0; p=0.0001) after controlling for patient age, duration of symptoms, radiographic appearance, pulmonary physiology, smoking history and sex. Honeycombing on HRCT indicated the presence of UIP with a sensitivity of 90% and specificity of 86%. Patients with nonspecific interstitial pneumonia were more likely to respond or remain stable (9 of 10) compared to patients with UIP (14 of 29) after treatment with prednisone. Patients remaining stable had the best prognosis. The risk ratio of mortality for stable patients compared to nonresponders was 0.32 (95% CI 0.11-0.93; p=0.04) in all patients and 0.33 (95% CI 0.12-0.96; p=0.04) in patients with UIP.The histological diagnosis of usual interstitial pneumonia is the most important factor determining survival in patients with suspected idiopathic interstitial pneumonia. The presence of honeycombing on high-resolution computed tomography is a good surrogate for usual interstitial pneumonia and could be utilized in patients unable to undergo surgical lung biopsy. Patients with nonspecific interstitial pneumonia are more likely to respond or remain stable following a course of prednisone. Patients remaining stable following prednisone therapy have the best prognosis. Eur Respir J 2002; 19: 275-283. This study was supported in part by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) Grant #P50HL46487, NIH/NCRR 3 MO1 RR00042-33S3, NIH/NIA P60 AG08808-06 and NHLBI, 1 K24 HL04212-01.A recent consensus statement has proposed that idiopathic interstitial pneumonias (IIPs) be divided into histopathological subsets that differ in prognosis and response to therapy [1]. These subsets include usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP), respiratory bronchiolitis interstitial lung disease (RBILD), desquamative interstitial pneumonia (DIP), and others [2,3]. This approach, by definition, requires surgical lung biopsy (SLB).The clinical features of IIP are nonspecific. Reliable noninvasive markers of the above patholog...

PTP-PEST is a ubiquitously expressed, cytosolic, mammalian protein tyrosine phosphatase (PTP) which exhibits high specific activity in vitro. We have investigated the substrate specificity of PTP-PEST by a novel substrate-trapping approach in combination with in vitro dephosphorylation experiments. We initially identified a prominent 130-kDa tyrosine-phosphorylated protein in pervanadate-treated HeLa cell lysates which was preferentially dephosphorylated by PTP-PEST in vitro. In order to identify this potential substrate, mutant (substrate-trapping) forms of PTP-PEST were generated which lack catalytic activity but retain the ability to bind substrates. These mutant proteins associated in stable complexes exclusively with the same 130-kDa protein, which was identified as p130(cas) by immunoblotting. This exclusive association was observed in lysates from several cell lines and in transfected COS cells, but was not observed with other members of the PTP family, strongly suggesting that p130(cas) represents a major physiologically relevant substrate for PTP-PEST. Our studies suggest potential roles for PTP-PEST in regulation of p130(cas) function. These functions include mitogen- and cell adhesion-induced signalling events and probable roles in transformation by various oncogenes. These results provide the first demonstration of a PTP having an inherently restricted substrate specificity in vitro and in vivo. The methods used to identify p130(cas) as a specific substrate for PTP-PEST are potentially applicable to any PTP and should therefore prove useful in determining the physiological substrates of other members of the PTP family.