RUOLO DI VAV1 NELLA MATURAZIONE DI PROMIELOCITI TUMORALI APL-DERIVATI

Vav1, one of the genetic markers that correlates with the differentiation of hematopoietic cells, belongs to a family of multidomain signal transduction proteins involved in the regulation of diverse cellular responses such as proliferation, differentiation, survival and migration. Normal and tumoral myeloid precursors are characterized by a low expression of this protein, that increases in amount and in tyrosine phosphorylation level during agonist-induced granulocytic differentiation. In blasts derived from acute promyelocytic leukemia (APL), the M3 subtype of acute myeloid leukemias, Vav1 plays a crucial role in the overcoming of the differentiation blockade. In particular, this protein promotes differentiation, potentiates ATRA-induced maturation and is involved in regulating expression of genes up-regulated by ATRA. It has also been demonstrated that in differentiating APL-derived cells Vav1 is tyrosine phosphorylated by Syk and this event is crucial for the maturation-related changes of cell morphology. Starting from these notions, the first aim of this study was to evaluate a possible role for Vav1 in modulating protein expression during ATRA-treatment of APL-derived cells. With this purpose, high-resolution 2-DE coupled with mass spectra analysis was performed on HL-60 and NB4 promyelocytes induced to differentiate with ATRA under conditions in which either the amount or the tyrosine phosphorylation of Vav1 were reduced. In both cell lines, the down-regulation of Vav1 affected the expression level of various proteins, including cell cycle/apoptosis- and cytoskeletonrelated proteins, that may constitute a common part of the ATRA-induced maturation pathway. These data indicate for Vav1 an unprecedented role in the differentiation of myeloid cells as a regulator of protein expression. A second aim of this work was to identify tyrosine residue(s) of Vav1 phosphorylated during ATRA treatment and involved in the overcoming of the differentiation blockade of APL-derived cells. Vav1 was then immunoprecipitated from differentiating NB4 cells and subjected to mass spectra analysis, allowing to identify the Y745 residue phosphorylated after ATRA treatment. The mutation of the residue Y745 of Vav1 impaired the completion of maturation program of NB4 cells, in terms of CD11b expression and migration capability. Since a functional role for Y745 has not been described so far, the tyrosine phosphorylation of this residue could be at the basis of a role of Vav1 alternative to the best known activity as guanosine exchange factor. As previously reported, the Syk-dependent tyrosine phosphorylation of Vav1 plays a role in the regulation of cell morphology of differentiating HL-60 cells. Since Vav1 interacts with Syk by means of its SH2 domain, where 4 tyrosines are present, the last part of this study was aimed to establish if these residues are possible substrates of Syk and to determine their functional role during ATRA-induced granulocytic differentiation. With this aim, HL-60 cells were transfected with plasmids containing mutated forms of Vav1 and induced to differentiate with ATRA. The role of each tyrosine residue on the differentiation process was studied by evaluating both CD11b expression and migration capability. The results indicate that both Y672 and Y711 are possible Syk substrates since their mutation, as well as Syk inhibition, have no effect on CD11b expression, but affect migration capability of ATRA-treated cells. The data reported in this work have considerably contributed to define the involvement of Vav1 in the maturation process of APL-derived cells, even though further analysis will be necessary to clearly establish its functional role.