Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells

Abstract

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.

Establishment and characterization of a novel imatinib‐sensitive chronic myeloid leukemia cell line MYL, and an imatinib‐resistant subline MYL‐R showing overexpression of Lyn

“…We next examined whether in vitro imatinib treatment was able to suppress the phosphorylated forms of CrkL. CrkL is a nuclear adaptor and transcriptional activator in BCR‐ABL‐expressing cells and constitutes the major substrate of BCR‐ABL in CML (35, 36). As shown, phosphorylation of CrkL was inhibited between 0.5 and 1 μ m imatinib, in both MYL and MYL‐R cells.…”

Crk is required for apoptosis in Xenopus egg extracts

“…It is tempting to speculate that the ability of bcr/abl to prevent apoptosis is mediated, at least in part, through the inhibitory phosphorylation of crk. In this regard, it should be noted that crkL, a protein very closely related to crk, is one of the major tyrosine‐phosphorylated proteins in chronic myelogenous leukemia cells expressing bcr/abl (Nichols et al ., 1994). It will be interesting to determine whether replacement of the endogenous crk protein with protein complexes containing a non‐phosphorylatable mutant of crk (the site of abl phosphorylation has been mapped to Tyr221 of crk) will impair the ability of abl to inhibit apoptosis.…”

Apoptosis in chronic myeloid leukemia cells transiently treated with imatinib or dasatinib is caused by residual BCR–ABL kinase inhibition

“…To prove that the TKI was completely removed from the system by a thorough washout, the restoration of BCR–ABL activity was demonstrated using phosphorylated CRKL (p‐CRKL) protein as a surrogate marker. The CRKL protein is a downstream signaling substrate of BCR–ABL, and its tyrosine phosphorylation serves as a specific indicator of BCR–ABL kinase activity in CML cells . Taken together, these data challenge the widespread notion that continuous BCR–ABL inhibition is required for the induction of apoptosis in CML cells by TKIs.…”