IDENTIFICATION OF CRKL AS THE CONSTITUTIVELY PHOSPHORYLATED 39-KD TYROSINE PHOSPHOPROTEIN IN CHRONIC MYELOGENOUS LEUKEMIA-CELLS

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic pr ecursors and their progeny. The Ph chromosome arises from a translocat ion that deregulates the c-ABL protein tyrosine kinase, giving it tran sforming 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. Thi s protein was prominently and constitutively tyrosine-phosphorylated i n CML neutrophils and was not phosphorylated in normal neutrophils. St imulation of normal neutrophils with cytokines and agonists did not in duce tyrosine phosphorylation of proteins migrating in the region of p p39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. Th e pp39 was not phosphorylated in hematopoietic cells from healthy dono rs or from patients with Ph chromosome-negative myeloproliferative dis orders. Using micro amino acid sequencing of purifled preparations of pp39, we identified pp39 as CRKL protein, which is consistent with rec ent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML c ells and cell lines as well as in antiphosphotyrosine immunoprecipitat es from CML cells. Our results suggest that pp39 CRKL in CML neutrophi ls may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an e arly stage of myeloid differentiation when the ABL kinase is active. C RK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signallin g 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 phosphorylatio n of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention. (C) 1994 by The American Society of Hema tology.