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W T Miller

Publications and source records attributed to W T Miller.

At least 37 records · Page 2Linked to original sources
PubMed2000

A peptide model system for processive phosphorylation by Src family kinases.

The Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src family kinases are involved in substrate recognition in vivo. Many cellular substrates of Src kinases contain a large number of potential phosphorylation sites, and the SH2 and SH3 domains of Src are known to be required for phosphorylation of these substrates. In principle, Src could phosphorylate these substrates by either a processive mechanism, in which the enzyme remains bound to the peptide substrate during multiple phosphorylation events, or a nonprocessive (distributive) mechanism, where each phosphorylation requires a separate binding interaction between enzyme and substrate. Here we use a synthetic peptide system to demonstrate that Hck, a Src family kinase, can phosphorylate substrates containing an SH2 domain ligand by a processive mechanism. Hck catalyzes the phosphorylation of these sites in a defined order. Furthermore, we show that addition of an SH3 domain to a peptide can enhance its phosphorylation both by activating Hck and by increasing the affinity of the substrate. On the basis of our observations on the role of the SH2 and SH3 domains in substrate recognition, we present a model for substrate targeting in vivo.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed2000

Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand.

The Src family tyrosine kinase Hck possesses two phosphorylation sites, Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K(m) value for peptide and a lower V(max) than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Amino Acid Motifs↗
PubMed2000

Distribution of thrombi in acute lower extremity deep venous thrombosis: implications for sonography and CT and MR venography.

OBJECTIVE: Our objective was to determine the typical distribution of thrombi in acute lower extremity deep venous thrombosis as a means of evaluating the validity of imaging techniques that only include the common femoral and popliteal veins, but not the superficial femoral vein. MATERIALS AND METHODS: The results of 2704 lower extremity venous sonograms, obtained in 2026 consecutive patients over a 4-year interval, were reviewed retrospectively. The distribution of acute deep venous thromboses across various lower extremity venous segments was analyzed for this population, which consisted of both symptomatic and asymptomatic patients. RESULTS: Of 2704 lower extremities studied with duplex sonography, acute deep venous thrombosis was identified in 269 (9.9%). Of these 269 cases, acute deep venous thrombosis was isolated to the superficial femoral vein in 60 (22.3%). The remaining 209 cases (77.7%) showed thrombus that extended into the common femoral or popliteal veins (or both). CONCLUSION: An abbreviated imaging study that evaluates only the common femoral and popliteal veins would fail to identify more than 20% of lower extremity acute deep venous thromboses in a population like ours. Although evaluation of the superficial femoral vein requires additional time and resources, evaluation of this segment may prevent a substantial number of thrombi from being missed.

Acute Disease↗
PubMed2000

CT findings in patients with esophagitis.

OBJECTIVE: The purpose of our study was to determine the CT findings in patients with esophagitis. MATERIALS AND METHODS: A search of medical and radiology files revealed 29 patients with esophagitis in whom thoracic CT was performed within 1 month of the diagnosis. The CT scans were reviewed retrospectively for esophageal wall thickening, a target sign, or other abnormalities. The thickness of the esophageal wall was also measured on CT in these 29 patients and compared with a control group of 85 patients. RESULTS: We found that 16 patients (55%) with esophagitis had abnormal findings on CT, including a thickened esophageal wall (using 5 mm as the threshold for wall thickening) in all 16 (55%) and a target sign in five (17%). The overall mean esophageal wall thickness was 4.7 mm (standard deviation [SD], 2 mm; 95% confidence interval [CI], 0.7-8.7 mm) in patients with esophagitis versus a mean wall thickness of 2.9 mm (SD, 0.8 mm; 95% CI, 1.3-4.5 mm) in controls (p <.001). Using the same 5-mm threshold for wall thickening, we found that only three (4%) of 85 controls had a thickened esophageal wall on CT. CONCLUSION: The majority of patients with esophagitis had abnormalities on CT, including a thickened esophageal wall (> or =5 mm) in 55% and a target sign in 17%. Although barium studies and endoscopy are more sensitive modalities for detecting this condition, the CT finding of a relatively long segment of circumferential esophageal wall thickening, with or without a target sign, should suggest the diagnosis of esophagitis in the proper clinical setting.

Adolescent↗
PubMed1999

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.

Circular Dichroism↗
PubMed1999

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 Acid Sequence↗
PubMed1999

Engineering the substrate specificity of the Abl tyrosine kinase.

c-Abl is a non-receptor tyrosine kinase that is involved in a variety of signaling pathways. Activated forms of c-Abl are associated with some forms of human leukemia. Presently, no high resolution structure of the tyrosine kinase domain of Abl is available. We have developed a structural homology model of the catalytic domain of Abl based on the crystal structure of the insulin receptor tyrosine kinase. Using this model as a guide, we selected residues near the active site predicted to play a role in peptide/protein substrate recognition. We expressed and purified 15 mutant forms of Abl with single amino acid substitutions at these positions and tested their peptide substrate specificity. We report here the identification of seven residues involved in recognition of the P-1, P+1, and P+3 positions of bound peptide substrate. Mutations in these residues cause distinct changes in substrate specificity. The results suggest features of Abl substrate recognition that may be relevant to related tyrosine kinases.

Amino Acid Sequence↗
PubMed1999

The x-ray plate.

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Forecasting↗
PubMed1999

Wither altruism.

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Career Choice↗
PubMed1999

Managed health care.

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Managed Care Programs↗
PubMed1999

Thickening of the diaphragm: a new computed tomography sign of diaphragm injury.

Computed tomography examination is becoming a standard method of evaluating injury in blunt trauma. This report describes a previously unreported computed tomography sign of diaphragmatic injury. A retrospective review of imaging findings was performed on eight patients with surgically proven traumatic diaphragmatic rupture. Thickening of the diaphragm was present on computed tomography in six of eight patients with surgically proven diaphragm rupture. Two patients demonstrated previously established computed tomography signs of diaphragm rupture and two patients had no findings to suggest diaphragm injury. Diaphragm thickening is associated with diaphragm rupture and may be a useful diagnostic sign in the evaluation of patients with blunt trauma.

Adult↗