PURPOSE: Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. METHODS: Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled coil domain (CC), and through a naturally occurring protein-protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a "protein switch" or attachment of 4 nuclear localization signals (NLSs). RESULTS: Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. CONCLUSIONS: We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.
PURPOSE:Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. METHODS:Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled coil domain (CC), and through a naturally occurring protein-protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a "protein switch" or attachment of 4 nuclear localization signals (NLSs). RESULTS: Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. CONCLUSIONS: We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.
Authors: Andrew S Dixon; Scott S Pendley; Benjamin J Bruno; David W Woessner; Adrian A Shimpi; Thomas E Cheatham; Carol S Lim Journal: J Biol Chem Date: 2011-06-09 Impact factor: 5.157
Authors: L Han; D Wong; A Dhaka; D Afar; M White; W Xie; H Herschman; O Witte; J Colicelli Journal: Proc Natl Acad Sci U S A Date: 1997-05-13 Impact factor: 11.205
Authors: Tomoyuki Tanaka; Helen Sewell; Simon Waters; Simon E V Phillips; Terence H Rabbitts Journal: J Biol Chem Date: 2010-10-27 Impact factor: 5.157
Authors: Jonathan E Constance; David W Woessner; Karina J Matissek; Mohanad Mossalam; Carol S Lim Journal: Mol Pharm Date: 2012-10-12 Impact factor: 4.939
Authors: Pamela Y Ting; Robert Damoiseaux; Björn Titz; Kenneth A Bradley; Thomas G Graeber; Virneliz Fernández-Vega; Thomas D Bannister; Peter Chase; Reji Nair; Louis Scampavia; Peter Hodder; Timothy P Spicer; John Colicelli Journal: PLoS One Date: 2015-03-26 Impact factor: 3.240