Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Identification of STAT3 as a specific substrate of breast tumor kinase.

Breast tumor kinase (Brk) is a non-receptor tyrosine kinase distantly related to the Src family kinase. It is expressed in more than 60% of breast tumors, but the biological role of this kinase remains to be determined. Only a limited number of substates have been identified for Brk, and the link of Brk to tumorigenesis remains largely unknown. In this study, we provide evidence that the signal transducer and activator of transcription 3, STAT3, is a physiological target of Brk. Activation of STAT3 previously has been linked to oncogenesis, and results in this study demonstrate that STAT3 is tyrosine phosphorylated and transcriptionally activated in cells expressing endogenous Brk. Signal transducer and activator of transcription 3 is specifically targeted since other STAT members are not responsive to Brk expression. Signal transducer and activator of transcription 3 activation requires the catalytic activity of Brk, and expression of both STAT3 and Brk stimulate cellular proliferation. In addition, we have identified a negative regulator of Brk, the suppressor of cytokine signaling, SOCS3. The SOCS3 protein is known to block signaling mediated by cytokine receptors, and here we find that SOCS3 is able to repress the activity of the Brk non-receptor tyrosine kinase.

Application of a human tumor colony-forming assay to new drug screening.

The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.

Protein phosphatase 2A interacts with the Src kinase substrate p130(CAS).

In this study, we report that the Src substrate Cas (p130 Crk-associated substrate) associates with protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We investigated this interaction in cells expressing a temperature-sensitive mutant form of v-Src. v-Src activation (by shifting cells from the nonpermissive to the permissive temperature) led to an increase in the tyrosine phosphorylation of v-Src and Cas, as well as in the association between v-Src and Cas. v-Src has previously been shown to bind to PP2A and to phosphorylate the catalytic subunit of PP2A, resulting in inhibition of phosphatase activity. We found that the association between v-Src and PP2A decreased as cells were shifted to the permissive temperature. In contrast, the levels of PP2A that co-immunoprecipitated with Cas increased when v-Src was activated. We obtained similar results in pull-down experiments with immobilized Microcystin, a PP2A inhibitor. Serine/threonine phosphorylation of Cas has previously been shown to occur in a cell cycle regulated matter. Treatment of NIH3T3 cells with okadaic acid, a PP2A inhibitor, augments the serine/threonine phosphorylation of Cas that occurs at mitosis. Furthermore, PP2A dephosphorylates serine residues on Cas in vitro. Taken together, our results suggest that PP2A may be involved in the cell cycle-specific dephosphorylation of Cas.

Autonomous folding of a C-terminal inhibitory fragment of Escherichia coli isoleucine-tRNA synthetase.

We previously reported that C-terminal fragments of Escherichia coli Ile-tRNA synthetase, a monomeric enzyme of 939 amino acids, act as dominant negative inhibitors of the wild-type enzyme in vivo and in vitro. Our experiments suggested that it is possible to block the functional assembly of a monomeric protein by interfering with the folding pathway. We postulated that the inhibitory C-terminal fragments fold autonomously, and in the presence of full-length Ile-tRNA synthetase, trap the N-terminal portion of polypeptide in an unproductive complex. Here, we report the results of experiments aimed at understanding the mechanism of dominant negative inhibition. We have carried out biophysical experiments on fragment 585-939 of Ile-tRNA synthetase, which we previously determined to be the minimal inhibitory unit. Circular dichroism and fluorescence spectroscopy indicate that this fragment forms a compact and stable structure in solution. The secondary structure of this fragment is predominantly alpha-helical, consistent with the crystal structure of Ile-tRNA synthetase from another organism. The C-terminal fragment is capable of forming native-like secondary and tertiary structure after refolding from guanidine HCl. Taken together, the results are consistent with the hypothesis that the inhibitory fragment of Ile-tRNA synthetase forms an independent folding unit.

Phosphorylation and activation of cGMP-dependent protein kinase by Src.

Using information obtained from experiments with peptide substrates of v-Src, a motif within the cGMP-binding domain of cGMP-dependent protein kinase (cGK) was identified as a potential phosphorylation site for v-Src. Here we show that the purified Ialpha isozyme of cGK is phosphorylated stoichiometrically and in a time-dependent manner by purified Src in vitro. The kinase activity of cGK is elevated approximately 4-fold (relative to autophosphorylated cGK) or 10-fold (relative to unphosphorylated cGK) upon tyrosine phosphorylation by Src. Phosphorylation of cGK by v-Src produces modest effects on the cGMP-binding properties and dissociation rates of cGK, and reduces the kact for cGMP. We hypothesize that the mechanism of activation may involve coupling of the cGMP binding domain to the catalytic domain.

Improving SH3 domain ligand selectivity using a non-natural scaffold.

BACKGROUND: Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS: We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS: Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk.

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.

Inhibition of Src by direct interaction with protein phosphatase 2A.

In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full-length v-Src or truncated forms of v-Src. The inhibition of v-Src by PP2A is concentration- and time-dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A-dependent inhibition of v-Src. When experiments were carried out at 4 degrees C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v-Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v-Src.

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.

Amino-terminal sequence determinants for substrate recognition by platelet-derived growth factor receptor tyrosine kinase.

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala-Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.

Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.

In this study, we report that the tyrosine kinase, Janus kinase 2 (Jak2), associates with the serine/threonine protein phosphatase 2A (PP2A) in 32Dcl3 myeloid progenitor cells. The association between Jak2 and PP2A transiently increases following interleukin-3 (IL-3) stimulation and activation of Jak2. The catalytic subunit of PP2A is tyrosine phosphorylated by Jak2 in vitro and in vivo, resulting in inhibition of phosphatase activity. PP2A also associates with Stat5 in 32Dcl3 cells in an IL-3-dependent manner. Pretreatment of 32Dcl3 cells with okadaic acid (OA), an inhibitor of PP2A, resulted in increased tyrosine phosphorylation and nuclear translocation of Stat5. Our results suggest that PP2A plays a negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with Jak2 and Stat5.

Role of fiberoptic bronchoscopy in the diagnosis of invasive pulmonary aspergillosis in patients with acute leukemia.

The utility and safety of fiberoptic bronchoscopy in the diagnosis of invasive pulmonary aspergillosis in patients with acute leukemia have not been examined. The results of 21 bronchoscopic procedures in 19 patients with invasive pulmonary aspergillosis and acute leukemia were reviewed. Analysis was confined to the 16 patients who had histopathologically documented infection on biopsy or at autopsy. Fiberoptic bronchoscopy established or suggested the diagnosis of invasive pulmonary aspergillosis in eight of 16 (50 percent) patients. Transbronchial or bronchial biopsy added only one diagnosis to those obtained by bronchial washing and brushing. Although fiberoptic bronchoscopy was a safe and well-tolerated procedure in our patients with invasive pulmonary aspergillosis and acute leukemia, its success rate was only 50 percent overall, and it appeared to be even less successful when performed early in the course of the disease. Fiberoptic bronchoscopy is a useful first procedure for the evaluation of patients with acute leukemia and possible invasive pulmonary aspergillosis, but a negative result does not exclude aspergillosis. Further diagnostic procedures, including repeated bronchoscopy, or institution of empiric antifungal therapy may be warranted if the clinical suspicion of invasive pulmonary aspergillosis is high.

Investigating the substrate specificity of the HER2/Neu tyrosine kinase using peptide libraries.

The product of the HER2/Neu oncogene is a receptor tyrosine kinase that is amplified in 25-30% of human primary breast tumors. In this project, we have isolated the HER2/Neu kinase from Sf9 cells infected with a baculovirus expression vector. We probed the substrate specificity of the HER2/Neu kinase using two peptide libraries: (1) a soluble peptide library containing three degenerate positions N-terminal to tyrosine; and (2) a bead-supported combinatorial library possessing six degenerate positions at P-1, P-2, P-3, P+1, P+2, and P+3. We identified four novel substrate sequences for HER2/Neu from the two peptide libraries. We synthesized these peptides as individual sequences and measured steady-state kinetic properties for phosphorylation by HER2/Neu. One of the peptides, AAEEIYAARRG, is the best synthetic peptide substrate reported to date for HER2/Neu. All of the sequences bear a resemblance to sites of autophosphorylation on HER2/Neu and related epidermal growth factor (EGF) receptor family tyrosine kinases.

Differential phosphorylation of IRS-1 by insulin and insulin-like growth factor I receptors in Chinese hamster ovary cells.

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are closely related receptor tyrosine kinases. Despite their high degree of homology, recent evidence suggests that the two receptors have distinct biological roles. In several recent studies, the cytoplasmic tyrosine kinase domains of the two receptors have been shown to possess different signalling specificities. In this study, we examine the hypothesis that differential phosphorylation of insulin receptor substrate 1 (IRS-1) may contribute to these differences in signalling between the two receptors. Using Chinese hamster ovary (CHO) cells stably expressing human IR or IGF-IR and activated by their respective ligands, we show that there are differences between the two receptors with regard to the complement of SH2-containing proteins recruited to IRS-1. In particular, IGF-IR appears to couple IRS-1 preferentially to Grb2 whereas, in contrast, IR appears to couple IRS-1 preferentially to the p85 subunit of phosphatidyl inositol 3-kinase (PI3-kinase) and to Nck. The two receptors couple IRS-1 equally to the tyrosine phosphatase SHP2. We have also generated phosphospecific antibodies to three important tyrosine phosphorylation sites on IRS-1 (pY608, pY895 and pY1172). We used these antibodies to probe the phosphorylation status of these sites in intact CHO/IR and CHO/IGF-IR cells. In the case of pY608, these results also show evidence for differential phosphorylation of IRS-1 by the two receptors. Taken together, the results presented here support the notion that the cytoplasmic domains of IR and IGF-IR have differences in their intrinsic signalling potentials.

Pulmonary aspergillosis in patients with AIDS. Clinical and radiographic correlations.

OBJECTIVE: To evaluate the clinical and radiographic features of pulmonary aspergillosis as they present in AIDS patients; in particular, to determine similarities and differences between Aspergillus infection in patients with AIDS vs those without AIDS. SUBJECTS AND METHODS: Six new cases of confirmed or probable pulmonary aspergillosis were discovered during a search of hospital records. These are reviewed with 30 previously reported cases with special attention to radiographic appearance of disease and how radiographic appearance influences clinical outcome. RESULTS: Symptoms of pulmonary aspergillosis in AIDS were nonspecific, most often including fever, cough, and dyspnea, and less commonly, chest pain or hemoptysis. Major risk factors for the development of pulmonary aspergillosis in patients with AIDS were steroid administration and neutropenia. Neutropenia was often a complication of therapies for AIDS, in particular, ganciclovir and zidovudine. Radiographic appearance of disease could be divided into three general categories. One third of the patients (13/36) presented with cavitary upper lobe disease resembling noninvasive or chronic necrotizing aspergillosis. Fatal hemoptysis occurred in 42 percent of patients with this form of disease. Twenty-two percent (8/36) of the cases presented as a nondescript focal alveolar opacity similar to invasive aspergillosis. In several patients, the focal infiltrate remained stable for several months, a feature that is unusual for aspergillosis in non-AIDS patients. The air crescent sign was present in none of the 36 reported cases. Patients with only focal disease had the best prognosis of patients with pulmonary aspergillosis. Bilateral alveolar or interstitial disease similar to invasive aspergillosis was present in 23 percent (9/36) of the patients. Bilateral disease appears to be a marker for disseminated infection and was associated with a high mortality due to aspergillosis. Two new forms of bronchial aspergillosis (5/36 cases) have been described previously. These patients presented with either obstructing fungal casts or bronchial pseudomembranes demonstrated bronchoscopically. In some patients with the bronchial forms of aspergillosis, transient alveolar opacities were seen on chest radiographs. These opacities may represent regions of atelectasis due to airway obstruction. One patient who had bilateral pneumothoraces without parenchymal opacities did not correspond to any of the three previously mentioned categories. Mortality due to aspergillosis was greater than 50 percent among AIDS patients. Death was subsequent to fatal hemoptysis or widespread pulmonary or systemic infection. CONCLUSION: Unlike other risk groups that tend to contract only one form of pulmonary aspergillosis, AIDS patients can develop the whole spectrum of aspergillosis-related pulmonary disorders, including chronic cavitary, invasive, and bronchial forms of aspergillosis. Clinical symptoms are nonspecific and major risk factors include neutropenia, which is often a side effect of various therapies for AIDS, and steroid administration. Patients with the chronic cavitary form of disease have an unusually high mortality due to fatal hemoptysis. Patients with bilateral pulmonary infiltrates and aspergillosis have a high mortality due to disseminated infection.

CD4 T lymphocyte count and the radiographic presentation of pulmonary tuberculosis. A study of the relationship between these factors in patients with human immunodeficiency virus infection.

BACKGROUND: Pulmonary infection and tumor in the AIDS population has a variable clinical and radiographic presentation. The association between the radiographic presentation of pulmonary tuberculosis and CD4 T lymphocyte count in the HIV-infected patient is investigated in order to provide an empirical approach for early diagnosis, treatment, and isolation of infected subjects. METHODS: A retrospective analysis of chest radiographs, CD4 T lymphocyte counts, and clinical history of 35 subjects from 3 urban hospitals was performed. All subjects were HIV-seropositive and had culture-proven pulmonary tuberculosis. Radiographs were evaluated for the presence of either a pattern characteristic of post-primary tuberculosis (typical pattern) or a pattern uncharacteristic of post-primary infection (atypical pattern). RESULTS: Twenty-one of 26 subjects with a CD4 T lymphocyte count less than 0.20 x 10(9) cells/L, whereas only 1 of 9 subjects with a CD4 T lymphocyte count of 0.20 x 10(9) cells/L or more presented with an atypical pattern of pulmonary tuberculosis (p < 0.001). The mean CD4 T lymphocyte counts of those subjects presenting with atypical versus typical radiographic pattern of post-primary pulmonary tuberculosis were 0.069 x 10(9) cells/L (n = 22) and 0.323 x 10(9) cells/L (n = 13), respectively (p < 0.01). Twenty-one of the 22 subjects with an atypical radiographic pattern of pulmonary tuberculosis were significantly immunosuppressed (CD4 < 0.20 x 10(9) cells/L). Atypical radiographic pattern included diffuse and lower lobar opacities, pleural effusion, mediastinal adenopathy, interstitial nodules, and a normal chest radiograph. CONCLUSION: AIDS patients presenting with CD4 count less than 0.20 x 10(9) cells/L and an atypical radiographic pattern for pulmonary tuberculosis are at risk for tuberculous infection requiring appropriate treatment and isolation until the diagnosis of pulmonary tuberculosis has been excluded.

Tuberculous pleural effusions.

While a number of recent reports have documented the changing clinical and radiographic spectrum of parenchymal tuberculosis, relatively little attention has been paid to changes in the patterns of pleural tuberculosis. We therefore reviewed the clinical, laboratory, and radiographic characteristics of 26 adult patients with tuberculous pleural effusions. We found that pleural tuberculosis has become a disease of older adults (median age, 56 years) and that 19 percent (5/26) of the cases were due to postprimary (reactivation) disease. This shift in age led to problems in diagnosis, since many of these older patients had underlying or coexisting disease that could have caused a pleural effusion. Both specimens of pleural fluid and pleural biopsy were useful in establishing the diagnosis. Examination of sputum was less helpful. All patients who were not anergic had positive cutaneous reactions to first-strength purified protein derivative of tuberculin. Lymphocytosis of the pleural fluid was not a uniform finding; only 62 percent of our patients had greater than 50 percent lymphocytes on their initial examinations of pleural fluid, and four patients had greater than 90 percent polymorphonuclear cells. All of the effusions were exudates, and four had glucose levels in the pleural fluid that were less than 30 mg/dl. Pleural tuberculosis is an important diagnostic consideration in adult or elderly patients with exudative pleural effusions.

Whole-lung tomography in urologic malignancy.

The place of whole-lung tomography in urologic malignancy has not been established. We reviewed 88 cases of known or suspected urologic malignancy in which tomography was used. Most patients had renal, bladder, or testicular tumors. Of the 68 patients with negative screening chest roentgenograms, all but 2 had negative tomograms. One of these 2 patients had a normal chest film (the other had extrapulmonary metastasis), and he did not have the renal carcinoma initially suspected. Tomography discovered no metastases from urologic malignancy that was not already known about from the screening x-ray film. In 20 cases with a positive screening x-ray film, tomography was of limited value. It cleared two suspicious chest films and added information on extent of metastatic involvement in three. We conclude that whole-lung tomography adds little information not available by chest roentgenogram in our selected population, and with a negative screening chest film no additional chest study is needed.