Detection in primary chronic myeloid leukaemia cells of p210BCR‐ABL1 in complexes with adaptor proteins CBL, CRKL, and GRB2

Abstract

Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR-ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR-ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR-ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL1, precluding accurate p210BCR-ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid-dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR-ABL1-degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR-ABL1 and cytoplasmic effector proteins in primary CML material.

Subcellular distribution of p210BCR-ABL in CML cell lines and primary CD34+ CML cells

“…Recently, we developed a method for inhibiting the proteolytic degradation by high-pH lysis that maintains protein integrity and allows IP studies of p145 ABL and p210 BCR-ABL in primary cells for the first time. 12 CT10 regulator like (CRKL) protein was first cloned by ten Hoeve in 1993 13 on chromosome 22. The gene encodes for a 39 kDa protein that is found most abundantly in haemopoietic tissue.…”

“…12 Levels of CRKL phosphorylation can be used as an indirect marker of p210 BCR-ABL function in response to therapy by western blot 19 and fluorescence-activated cell sorting assay. 20 Many questions remain about the composition of the protein complexes in primary CML cells.…”

Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells

“…[1][2][3] The mechanisms responsible for the transition of CML CP into BC remain poorly understood, although it is generally accepted that an unrestrained BCR-ABL activity and accumulation of additional genetic alterations in hematopoietic stem/ progenitor cells are the primary determinants for expansion of increasingly malignant cell clones. [4][5][6] Molecular changes commonly observed in evolution to BC are oncogene mutations as well as additional nonrandom chromosomal abnormalities involving whole chromosomes. 3,7,8 Such massive genomic changes, collectively known as aneuploidy, are characteristic features of human tumors and contribute to the multistep model of tumorigenesis.…”

Antibody arrays identify protein‐protein interactions in chronic myeloid leukaemia

“…Samples were centrifuged at 10 000 g for 1 min. p210 BCR-ABL1 from CML cell lines was immunoprecipitated with ExactaCruz TM (Santa Cruz Biotechnology), prepared as previously described (Patel et al, 2006).…”

“…As a result, the core complex contains a tyrosine phosphatase domain in Sts-1 and the 5-inositolphosphatase domain in SHIP-2, raising the possibility that targeting these enzyme activities in addition to the tyrosine kinase activity of p210 BCR-ABL1 may be useful therapeutically. It is relevant that treatment with tyrosine kinase inhibitors reduces the association with p210 BCR-ABL1 of some proteins (c-Cbl, Crk-1 and Crk-L) more than others (Grb-2) (Patel et al, 2006;Brehme et al, 2009). Moreover, a BCR-ABL1 mutant lacking binding sites for Grb-2, c-Cbl and Crk-L is unable to induce leukaemia in mice (Johnson et al, 2009).…”