Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gainor loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.Bcr-Abl, a constitutively active tyrosine kinase, is the defining molecular feature of chronic myeloid leukemia (CML) (8). Biochemical studies and murine models have established that tyrosine kinase activity is essential to the transforming capacity of Bcr-Abl (7, 17). Consistent with this, inhibition of the BcrAbl kinase with imatinib, an Abl-specific kinase inhibitor, induces remissions in patients with CML (9, 10). Although responses in the early phases of CML are frequently durable, relapse is common in patients with advanced-phase CML. Mutations in the kinase domain (KD) of Bcr-Abl that impair imatinib binding have been identified as the major mechanism of acquired resistance (1,3,13,15,34,37). However, in some patients, drug-resistant mutants were detected prior to therapy (16,27,28,34,38). The proportion of mutant allele in these samples varied, from being detectable only with allele-specific PCR, which could detect as few as one cell in 100,000 (38), to representing 40% of the BCR-ABL message (34). These data suggest that some mutants may have a proliferative advantage over unmutated Bcr-Abl (herein referred to as native Bcr-Abl) even in the absence of imatinib, while others may be loss-offunction alleles that are selected only in the presence of imatinib. Mutations of the ATP binding loop (P-loop) of Abl are associated with significantly shorter survival than other mutations, regardless of their sensitivity to imatinib (3, 36), suggesting that certain Bcr-Abl mutations may directly contribute to disease progression by conferring a more aggressive phenotype. To test these hypotheses, we performed various biochemical and biological assays to compare five common KD mutants that comprise ca. 60% of Bcr-Abl m...

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Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored by nuclear magnetic resonance (NMR) techniques. The chemical shift perturbation data show the probable contacts that CheW makes with CheA. In combination with previous genetic data, the structure also suggests a possible binding site for the chemotaxis receptor. These results provide a structural basis for a model in which CheW acts as a molecular bridge between CheA and the cytoplasmic tails of the receptor.