Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in chronic myelogeous leukemia. Although crystal structures of the down-regulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to smallmolecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was used to compare the conformations of wild-type Abl with a nonmyristoylated form and with 3 clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the down-regulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function. allosteric interactions ͉ chronic mylogenous leukemia ͉ hydrogen exchange ͉ mass spectrometry

The Bcr-Abl kinase inhibitor imatinib is remarkably effective in chronic myelogenous leukemia (CML), although drug resistance is an emerging problem. Myeloid Src family kinases such as Hck and Lyn are often overexpressed in imatinib-resistant CML cells that lack Bcr-Abl mutations. Here we tested whether Hck overexpression is sufficient to induce imatinib resistance using both wild-type Hck and a mutant (Hck-T338A) that is uniquely sensitive to the pyrazolopyrimidine inhibitor, NaPP1. Expression of either kinase in K562 CML cells caused resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in cells expressing T338A but not wild-type Hck, demonstrating that resistance requires Hck kinase activity. NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib sensitivity exclusively in cells expressing Hck-T338A. These data show that elevated Src family kinase activity is sufficient to induce imatinib resistance through a mechanism that may involve phosphorylation of Bcr-Abl. Chronic myelogenous leukemia (CML)2 is a hematopoietic stem cell disease with three clinical phases. The initial chronic phase involves expansion of myeloid cells that retain their ability to undergo terminal differentiation. As the disease progresses, patients enter an accelerated phase followed by a blast crisis characterized by differentiation arrest and accumulation of immature blast cells in the bone marrow and peripheral blood (1).The cytogenetic hallmark of CML is the Philadelphia chromosome, which fuses the BCR (breakpoint cluster region) locus on chromosome 22 with the c-ABL proto-oncogene on chromosome 9. This translocation is present in greater than 90% of CML patients and leads to the expression of Bcr-Abl, a chimeric protein of 210 kDa (2, 3) with abnormal subcellular localization and constitutive protein-tyrosine kinase activity (4, 5). Bcr-Abl drives the pathogenesis of CML through the phosphorylation and activation of a broad range of downstream signaling proteins that increase cell survival and promote unregulated cell cycle progression (6). These pathways include but are not limited to the Ras/mitogen-activated protein kinase (MAPK), NF-B, phosphatidylinositol 3-kinase/Akt, and Stat signaling cascades (7-9).Bcr-Abl has been shown to activate other non-receptor protein-tyrosine kinases, particularly Src family kinases (SFKs) expressed in myeloid cells such as Hck and Lyn (10). A growing body of evidence supports the relevance of this interaction to CML pathogenesis. For example, expression of a kinase-defective mutant of Hck blocked Bcr-Abl-induced transformation of murine myeloid leukemia cells to cytokine independence (11). In addition, Hck was shown to couple Bcr-Abl to Stat5 signaling and to be required for Bcr-Abl-induced transformation of murine myeloid cells (12). Furthermore, SFK-selective inhibitors block proliferation and induce apoptosis in CML cells without affecting Philadelphia chromosome-negative...

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BackgroundNef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.ResultsTo test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.ConclusionsThese observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

Chronic Myelogenous Leukemia (CML) is characterized by the presence of Philadelphia Chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates signaling pathways important for the proliferation, survival and cytokine independence of myeloid progenitors, including the Src family kinases (SFKs). Imatinib, an inhibitor of Bcr-Abl tyrosine kinase activity, is the frontline therapy for CML. Although imatinib is remarkably effective in the chronic phase of the disease, patients with accelerated or blast crisis CML often develop resistance. Several recent studies performed on clinical specimens from imatinib-resistant patients with wild-type Bcr-Abl found that the Src family kinase members Hck and Lyn are overexpressed or highly active, suggesting that Src kinases may play a role in imatinib resistance. While the role of Lyn has been addressed in some detail, the contribution of Hck to this type of imatinib resistance is less well understood. To test whether Hck overexpression in CML cells induces resistance to imatinib in a kinase-dependent manner, we employed a Hck mutant (Hck-T338A) that is uniquely sensitive to the pyrazolo-pyrimidine inhibitor, NaPP1. In vitro, Hck-T338A was 48 times more sensitive to NaPP1 than the wild-type kinase. Importantly, this “gatekeeper” mutation was functionally silent and did not confer a loss or gain of function on Hck in a control fibroblast transformation assay. Expression of wild-type Hck or the T338A mutant in K562 CML cells resulted in resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in a concentration-dependent manner only in cells expressing the Hck-T338A mutant. In contrast, cells expressing wild-type Hck were not affected by NaPP1, demonstrating the selectivity of NaPP1 for the Hck-T338A mutant. This result shows that Hck-induced imatinib resistance requires Hck kinase activity. In addition, NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib binding exclusively in cells expressing Hck-T338A. Together, these data establish a direct cause and effect relationship between Hck overexpression and imatinib resistance in CML cells. Furthermore, our results show that imatinib resistance requires Hck kinase function and may occur via direct Hck-induced trans-phosphorylation of Bcr-Abl. Selective drug targeting of Hck may be of therapeutic benefit in imatinib-resistant CML patients with increased Hck expression or activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3687.