Mass spectrometry-based identification of Y745 of Vav1 as a tyrosine residue crucial in maturation of acute promyelocytic leukemia-derived cells.

Vav1, whose physiological expression is restricted to hematopoietic system, is one of the signaling proteins up-regulated by all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL)-derived precursors, in which it promotes the overcoming of the differentiation blockade. High levels of tyrosine phosphorylated Vav1 accumulate in differentiating APL-derived cells, suggesting that one or more Vav1 tyrosine residues are involved in neutrophil differentiation of tumoral promyelocytes. Here, we have found that phosphorylation of Vav1 Y174, that is known to regulate Vav1 activity in mature neutrophils, is up-regulated by ATRA in NB4 cells. Nevertheless, this tyrosine residue does not seem crucial for the agonist-induced phenotypical differentiation of APL-derived cells. Mass spectrometry analysis performed on Vav1 from differentiating NB4 cells allowed to identify the highly conserved Y745 residue as a phosphorylated tyrosine that plays crucial roles in the completion of the maturation program of this cell line. In fact, the overexpression of a mutated form of Vav1, in which Y745 was replaced with a phenylalanine, significantly reduced the ATRA-induced CD11b expression and essentially abrogated the differentiation-related acquisition of the migratory capability. Even though the intracellular signaling involving Vav1 phosphorylated in Y745 is unknown, the identification of a tyrosine residue essential for differentiation of tumoral precursors may constitute the basis to identify new specific targets for differentiation therapy of APL.