C-CBL TYROSINE PHOSPHORYLATION AND SUBCELLULAR-LOCALIZATION IN HUMAN PRIMARY LEUKEMIC-CELLS

Several studies indicate that a number of signal-transducing molecules involved in the proliferation, differentiation, and functional activa tion of normal hemopoietic cells may be constitutively activated in pr imary leukemic cells and play a role in the outcome or in the progress ion of these neoplastic disorders. In this study we show that the prod uct of the proto-oncogene c-Cbl, whose function is still unknown, is c onstitutively tyrosine phosphorylated not only in cells from chronic m yelogenous leukemias (CMLs) in the blast phase, but also in cells from acute myeloblastic leukemias (AMLs), Ph-negative acute T-lymphoblasti c leukemias (T-ALLs), and Ph-negative pre-B lymphoblastic leukemias (p re-B ALL). Moreover, in acute leukemia cells, c-Cbl was not stably com plexed with the tyrosine-phosphorylated adaptor protein CrkL. The anal ysis of Grb2/c-Cbl interaction demonstrated that, in both acute leukem ia and CML blasts, c-Cbl was stably complexed with the N-terminal Src homology (SH) 3 domain of Grb2 and, in blasts from ALL patients, with the Grb2 SH2 domain. The analysis of c-Cbl subcellular distribution sh owed that in all cases of leukemia tested, as well as in growth factor -stimulated M-07e cells, c-Cbl was present in the cytosolic, in the me mbrane, and in the detergent-insoluble fractions. Finally, in polymorp honuclear neutrophils (PMNs) from CML patients, c-Cbl was found stably associated with the detergent-insoluble fraction, whereas in PMNs fro m normal donors, it was detected only in the cytosolic fraction. Our f indings that c-Cbl is constitutively tyrosine phosphorylated and assoc iated with the detergent-insoluble fraction in AML and ALL blasts and in PMNs from CML patients suggest that this event represents a common step in the neoplastic transformation of both myeloid and lymphoid pro genitor cells.