All-trans-retinoic acid induces tyrosine phosphorylation of the CrkL adapter in acute promyelocytic leukemia cells

Objective. All-trans-retinoic acid (RA) is a potent inducer of differentiat ion of acute promyelocytic leukemia (APL) cells in vitro and in vivo. It al so exhibits synergistic effects with interferons an the induction of differ entiation and growth inhibition in vitro. Recent studies showed that interf erons engage a signaling pathway involving the CBL proto-oncogene and the C rkL adapter, which mediates interferon-induced growth inhibitory signals. T he objective of this study was to determine whether the CBL-CrkL pathway is activated by treatment of the NB-4 and HL-60 acute leukemia cell lines wit h RA. Materials and Methods. The effects of RA treatment on CBL and CrkL phosphor ylation, as well as on protein-protein interactions, were determined in stu dies involving immunoprecipitations of cell extracts with specific antibodi es and Western blots. in addition, glutathione-S-transferase fusion protein s were used in binding studies to determine whether the SH2 domain of CrkL interacts with CBL in a RA-dependent manner and whether Rap1 is activated b y RA. Results. Treatment of NB-I or HL-60 cells with Ri resulted in strong tyrosi ne phosphorylation elf CBL, which was time and dose dependent. Similarly, R A induced tyrosine phosphorylation of the CrkL adapter and the association of CrkL with CBL. The RA-dependent interaction of CrkL with CBL was mediate d by binding of the SH2 domain of CrkL to tyrosine phosphorylated CBL, sugg esting that CBL provides a docking site for engagement of CrkL in a RA-acti vated cellular pathway, The guanine exchange factor C3G was found to he ass ociated with CrkL at similar levels before and after RA treatment, but Rap1 activation downstream of C3G was nut inducible by RA. Conclusions. These findings demonstrate that the CBL-CrkL pathway is one of the mediators of the effects of RA on APL cells and suggest that one of th e mechanisms of synergy between RA and interferons may involve regulation o f components of this signaling cascade. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.