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N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.

ABSTRACT: Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

SUBMITTER: Choi Y

PROVIDER: S-EPMC2781447 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.

Choi Yongmun Y Seeliger Markus A MA Panjarian Shoghag B SB Kim Hakjoong H Deng Xianming X Sim Taebo T Couch Brian B Koleske Anthony J AJ Smithgall Thomas E TE Gray Nathanael S NS

The Journal of biological chemistry 20090813 42

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more p ...[more]

PMID: 19679652

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Project description:BACKGROUND: Point mutations of the BCR-ABL tyrosine kinase domain are considered the predominant cause of imatinib resistance in chronic myeloid leukemia. The expansion of mutant BCR-ABL-positive clones under selective pressure of tyrosine kinase inhibition is referred to as clonal selection; there are few data on the reversibility of this phenomenon. DESIGN AND METHODS: The changes of expression of mutant BCR-ABL-positive alleles after cessation of tyrosine kinase inhibitor treatment were examined in 19 patients with chronic myeloid leukemia harboring different mutations in a longitudinal follow-up. The proportion of mutant alleles was quantified by amplification of rearranged ABL sequences followed by mutation-specific restriction digestion, electrophoresis and densitometry. The size of mutant clones was established as a measure of the absolute amount of mutant cells considering the proportion of mutant BCR-ABL transcripts and the total level of BCR-ABL obtained by quantitative reverse transcriptase polymerase chain reaction. RESULTS: The median proportion of mutant transcripts was 97% before and 8% after cessation of tyrosine kinase inhibitor treatment indicating a relative decline of 88% within a median of 6 months. The relative decrease in the size of the mutant clones was 86%. Repeated selection and deselection of the mutant clone after resumption and second cessation of tyrosine kinase inhibitor treatment was observed in individual patients. CONCLUSIONS: Deselection of mutant BCR-ABL-positive clones after cessation of tyrosine kinase inhibitor treatment might be a common, rapid and reproducible phenomenon, although some patients harboring the T315I mutation showed no deselection. Cessation of tyrosine kinase inhibitor treatment may lead to the regression of T315I mutant clones to a level under the limit of detection, offering the therapeutic option of resumed tyrosine kinase inhibitor treatment under close surveillance of the mutation status.

Project description:Plasmodium falciparum extensively remodels human erythrocytes by exporting hundreds of parasite proteins. This remodeling is closely linked to the Plasmodium virulence-related functions and immune evasion. The N-terminal export signal named PEXEL (Plasmodium export element) was identified to be important for the export of proteins beyond the PVM, however, the issue of how these PEXEL-positive proteins are transported and regulated by Rab GTPases from the endoplasmic reticulum (ER) to the cell surface has remained poorly understood. Previously, we identified new aspects of the trafficking of N-myristoylated adenylate kinase 2 (PfAK2), which lacks the PEXEL motif and is regulated by the PfRab5b GTPase. Overexpression of PfRab5b suppressed the transport of PfAK2 to the parasitophorous vacuole membrane and PfAK2 was accumulated in the punctate compartment within the parasite. Here, we report the identification of PfRab5b associated proteins and dissect the pathway regulated by PfRab5b. We isolated two membrane trafficking GTPases PfArf1 and PfRab1b by coimmunoprecipitation with PfRab5b and via mass analysis. PfArf1 and PfRab1b are both colocalized with PfRab5b adjacent to the ER in the early erythrocytic stage. A super-resolution microgram of the indirect immunofluorescence assay using PfArf1 or PfRab1b- expressing parasites revealed that PfArf1 and PfRab1b are localized to different ER subdomains. We used a genetic approach to expresses an active or inactive mutant of PfArf1 that specifically inhibited the trafficking of PfAK2 to the parasitophorous vacuole membrane. While expression of PfRab1b mutants did not affect in the PfAK2 transport. In contrast, the export of the PEXEL-positive protein Rifin was decreased by the expression of the inactive mutant of PfRab1b or PfArf1. These data indicate that the transport of PfAK2 and Rifin were recognized at the different ER subdomain by the two independent GTPases: PfAK2 is sorted by PfArf1 into the pathway for the PV, and the export of Rifin might be sequentially regulated by PfArf1 and PfRab1b.

Dataset Information

N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.

Publications

N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor.

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