CRKL but not CRKII Inhibits Erythropoiesis and Megakaryopoiesis of CML via Inactivating Raf/MEK/ERK/Elk-1 Pathway

Abstract

Background: As members of the CT10 regulation of kinase (CRK) adaptor protein family, CRK-like (CRKL) and CRKII are involved in cell proliferation, survival, adhesion, migration and differentiation. However, the exact role and underlying mechanism of CRKL and CRKII in leukemic cell differentiation are still unknown. Methods: Quantitative real-time qPCR (qRT-PCR) was used to detect the expression levels of CRKL and CRKII in chronic myeloid leukemia (CML) patients and complete remission (CR) patients; Western blotting (WB) was used to measure the expression levels of CRKL and CRKII during hemin-induced erythroid differentiation of K562 cells; Benzidine staining, isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis, cDNA microarray assay, qRT-PCR and WB were used to examine the effects of CRKL and CRKII deregulation on erythroid and megakaryocyte differentiation of K562 cells; PD98059 was used to investigate the underlying mechanism of CRKL in erythropoiesis and megakaryopoiesis. Results: CRKL was found to be overexpressed in chronic myeloid leukemia (CML) patients compared with normal samples, while its expression level was lower in CR patients than in corresponding CML patients. The CRKL expression level was significantly decreased during the erythroid differentiation of K562 cells following hemin treatment. Moreover, CRKL downregulation promoted erythroid and megakaryocyte differentiation of K562 cells accompanied by increased expression level of the erythroid differentiation markers γ-globin, glycophorin (GPA) and the megakaryocyte differentiation markers CD41, CD61. Furthermore, gene microarray and iTRAQ quantitative proteomic analysis showed that CRKL downregulation increased hemoglobin (HB) molecules HBD, HBA1, HBA2, HBZ, HBE1, HBG1 and globin transcription factor 1 (GATA1), high-mobility group protein (HMGB2) expression levels. Mechanistically, CRKL inhibited erythroid and megakaryocyte differentiation of K562 cell via inactivating Raf/MEK/ERK/Elk-1 pathway. Conversely, CRKII was only slightly overexpressed in CML patients and had no effect on erythroid differentiation of K562 cells. Conclusions: Taken together, our results demonstrate that CRKL but not CRKII contributes to development, progression, erythropoiesis and megakaryopoiesis of CML, providing novel insights into effective diagnosis and therapy for CML patients.