Activation of the Axl receptor tyrosine kinase induces mitogenesis and transformation in 32D cells.
axl is a transforming receptor tyrosine kinase isolated from DNA of patients with chronic myelogenous leukemia. Association of axl expression with myelogenous leukemias and its expression in primitive hematopoietic cells suggests a role for axl in myeloid biology. To study the cellular function of axl, we constructed a chimeric receptor tyrosine kinase composed of the extracellular and transmembrane domains of the EGF receptor and the cytoplasmic domain of axl; this chimera was named EAK for EGFR-Axl-Kinase. The EAK chimeric receptor was expressed in the mouse myeloid progenitor cell line 32D, which is dependent on interleukin 3 (IL-3) for proliferation and survival. Treatment of the 32D-EAK cells with EGF stimulated the tyrosine phosphorylation of the axl kinase domain and enabled proliferation through EGF rather than IL-3. Thus, axl can effectively couple with mitogenic signaling pathways intrinsic to 32D myeloid cells. Assay of proteins phosphorylated in response to different cytokine treatments showed that IL-3 and EGF exposure produced unique profiles in the 32D-EAK cells. Furthermore, Jak-2 is phosphorylated only in response to IL-3 treatment in these cells. This suggests that IL-3 receptor and axl transduce mitogenic signals through separate pathways. In addition, exposure of cells expressing the chimeric receptor to EGF for 19 days converted the cells to factor-independent growth, a phenomenon not seen with other receptor tyrosine kinases. Generation of this transformed phenotype is absolutely dependent on axl activation by foster ligand. The tyrosine phosphorylation level of the axl kinase domain in the factor-independent subclones is 40-fold greater than the factor-dependent cells. The association of a unique axl phosphorylation level with the factor-independent phenotype suggests that there is a threshold phosphorylation level of the axl kinase for transformation. The fact that activation of the axl receptor leads to transformation of 32D cells suggests that axl can play a role in leukemic conversion of myeloid cells, either through inappropriate expression or improper activation.
Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599.
P
P
McCloskey
J
J
Pierce
R A
RA
Koski
B
B
Varnum
E T
ET
Liu
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology; D016922:Cellular Senescence / Q000502:physiology; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D000094963:Axl Receptor Tyrosine Kinase
D008934:Mitogens; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D000094963:Axl Receptor Tyrosine Kinase