Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl

Abstract

A novel C3G isoform, designated p87C3G, lacking the most amino terminal region of the cognate protein has been found to be overexpressed in two CML cell lines, K562 and Boff 210, both expressing Bcr-Abl p210. p87C3G expression is also highly augmented in patients diagnosed with chronic myeloid leukemia (CML) Ph+, in comparison with healthy individuals, and returns to basal levels after treatment with STI571. p87C3G co-immunoprecipitates with both CrkL and Bcr-Abl in CML cell lines and co-immunoprecipitation between p87C3G and Bcr-Abl was also detected in primary cells from CML patients. These interactions have been confirmed by in vitro pull down experiments. The interaction between p87C3G and Bcr-Abl involves the SH3-binding domain of p87C3G and the SH3 domain of Abl and depends mostly on the first polyproline region of p87C3G. Furthermore, we also demonstrated that p87C3G is phosphorylated in vitro by a Bcr-Abl-dependent mechanism. These results indicate that p87C3G overexpression is linked to CML phenotype and that p87C3G may exert productive functional interactions with Bcr-Abl signaling components suggesting the implication of this C3G isoform in the pathogenesis of chronic myeloid leukemia.

F-actin-binding domain of c-Abl regulates localized phosphorylation of C3G: role of C3G in c-Abl-mediated cell death

“…In Bcr-Abl-expressing leukaemic cells, phosphorylation was seen on p87C3G, a splice variant and not on fulllength C3G (Gutierrez-Berzal et al, 2006). Our results implicate differences in c-Abl and Bcr-Abl to engage and phosphorylate C3G.…”

NADPH Oxidases as Therapeutic Targets in Chronic Myelogenous Leukemia

“…K562, a human cell line derived from a patient with CML, was obtained from the ATCC (CCL 243). Boff 210 cells express p210 BCR-ABL constitutively and derive from the BaF/3 murine hematopoietic cell line, as previously described (24). Authentication of BM-MNC was performed analyzing BCR-ABL expression by PCR, or BCR-ABL protein expression in K562 and Boff 210 cells by immunoblotting, every 3 to 4 months.…”

Immunophilins Control T Lymphocyte Adhesion and Migration by Regulating CrkII Binding to C3G

“…The pGST-C3G and pGST-SH3b were gifts of C. Guerrero (Universidad de Salamanca-Consejo Superior de Investigaciones Cientificas, Salamanca, Spain) (17). GSTisomerase vectors were gifts of J. Bolstad and W. Chen (University of Calgary, Calgary, AB, Canada) (human [h]FKBP12 and hFKBP12.6), T. Ratajczak (University of Western Australia, Crawley, WA, Australia) (hFKBP51 and hCyp40), B. Chambraud (INSERM U488, Paris, France) (hFKBP52), M. Emerman (University of Washington, Seattle, WA) (hCypA), and J. Buchner (University of Technology, Munich, Germany) (hCyp40).…”

“…To distinguish between these two potential mechanisms, we precultured Jurkat T cell lysates for 24 h in the presence or absence of immunophilin inhibitors, CsA and/or FK506, and pulled down binding proteins using bead-immobilized GST-C3G or GST-SH3b [a truncated C3G corresponding to aa 208-662, which possesses multiple copies of proline-rich regions (17,33)] fusion proteins. Immunoblot analysis revealed that each of the two fusion proteins, GST-C3G and -SH3b, pulled down both CrkI and CrkII (Fig.…”