Vav1 Modulates Protein Expression During ATRA-Induced Maturation of APL-Derived Promyelocytes: A Proteomic-Based Analysis

Abstract

Overexpression of Vav1 promotes the overcoming of the differentiation blockade that characterizes acute promyelocytic leukemia cells. At variance, down-modulation of Vav1 prevents ATRA-induced maturation, and in particular, the inhibition of its tyrosine phosphorylation prevents the neutrophil differentiation-related changes of cell morphology. These findings allowed to identify Vav1 as a crucial protein in the ATRA-dependent differentiation of tumoral promyelocytes. By means of a proteomic approach, here we have investigated a possible role for Vav1 in modulating protein expression during ATRA treatment of tumoral promyelocytes. We have performed high-resolution 2-DE coupled with mass spectra analysis of HL-60 and NB4 promyelocytic cell lines induced to differentiate with ATRA when the amounts or the tyrosine phosphorylation of Vav1 were forcedly reduced. We have found that the down-regulation of Vav1 affects the expression level of a number of proteins, including cell cycle/apoptosis- and cytoskeleton-related proteins. In particular, the expression of 14-3-3epsilon, alpha-enolase, alpha-tubulin and splice isoform 2 of alpha3 proteasome subunit changed as a consequence of the down-modulation of Vav1 during the differentiation of both HL-60 and NB4 cell lines, suggesting that these proteins may constitute a common part of the ATRA-induced pathway during maturation of APL-derived promyelocytes. These results indicate an unprecedented role for Vav1 in the maturation of myeloid cells as a regulator of protein expression.

Vav1: A Key Player in Agonist-Induced Differentiation of Promyelocytes from Acute Myeloid Leukemia (APL)

“…The Syk-dependent tyrosine phosphorylation of Vav1 during the ATRA-induced phenotypical differentiation is not relevant for the expression of the surface marker CD11b, as indicated by the use of Piceatannol in both HL-60 and NB4 cells, but seems to play a crucial role in regulating the reorganization of cell architecture. In fact, when Piceatannol is administered in combination with ATRA, the modifications of nuclear morphology typical of granulocytic differentiation are almost completely abrogated, similarly to what observed when the expression of Vav1 is down-modulated during the differentiation treatment (Bertagnolo et al, 2008). However, since Piceatannol fails to abrogate completely the ATRA-induced tyrosine phosphorylation of Vav1 in both HL-60 and NB4 cells (Bertagnolo et al, 2008), other kinase/s, in addition to Syk, are probably recruited by ATRA in these cell models.…”

“…In fact, when Piceatannol is administered in combination with ATRA, the modifications of nuclear morphology typical of granulocytic differentiation are almost completely abrogated, similarly to what observed when the expression of Vav1 is down-modulated during the differentiation treatment (Bertagnolo et al, 2008). However, since Piceatannol fails to abrogate completely the ATRA-induced tyrosine phosphorylation of Vav1 in both HL-60 and NB4 cells (Bertagnolo et al, 2008), other kinase/s, in addition to Syk, are probably recruited by ATRA in these cell models. The results from these inhibition studies support the hypothesized action model that r e q u i r e s t y r o s i n e p h o s p h o r y l a t e d V a v 1 i n maturation of tumoral myeloid precursors.…”

“…While in whole HL-60 cells the Vav1/Syk association takes place regardless of their phosphorylation level and of ATRA treatment, the formation of Vav1/Syk complexes inside the nuclear compartment strongly increases during the differentiation process, suggesting a specific role for this tyrosine kinase in the nucleus. The role of Syk in phosphorylating Vav1 has been demonstrated by means of in vitro assays (Bertagnolo et al, 2001) and confirmed by the use of a pharmacological model of Syk inhibition, in which both HL-60 and NB4 cells were treated with Piceatannol (Bertagnolo et al, 2001(Bertagnolo et al, , 2008, a tyrosine kinase inhibitor with a reported selectivity for Syk (Law et al, 1999;Seow et al, 2002). The Syk-dependent tyrosine phosphorylation of Vav1 during the ATRA-induced phenotypical differentiation is not relevant for the expression of the surface marker CD11b, as indicated by the use of Piceatannol in both HL-60 and NB4 cells, but seems to play a crucial role in regulating the reorganization of cell architecture.…”

“…On the basis of the evidence that inside the nucleus of a number of different cell lines, including APL-derived cells, Vav1 participates to molecular complexes with DNA-related proteins Houlard et al, 2002;Romero et al, 1998) a more general role of Vav1 in regulating events ended to control protein expression can be hypothesized. 2D electrophoresis followed by mass spectrometry have established that, in both HL-60 and NB4 cells, the down-modulation of Vav1 abrogates the capacity of ATRA of modulating the expression of proteins associated to cytoskeleton and involved in proliferation and of apoptosis-related proteins, as well as of molecules implicated in metabolism, synthesis, folding and degradation of proteins (Bertagnolo et al, 2008). The majority of the identified proteins are affected by Vav1 down-modulation only in one of the two analyzed cell lines, according to the notion that HL-60 and NB4 cells, even if both derived from patients with APL, show peculiar genotypic and phenotypic profiles (Barber et al, 2008).…”

“…The use of siRNAs specific for Vav1 unequivocally demonstrates that Vav1 is not dispensable for the progression of tumoral promyelocytes along the granulocytic lineage. In fact, the down-modulation of Vav1 expression during ATRA treatment of both HL-60 and NB4 cells counteracts the agonist-induced increase of CD11b expression and prevents the maturation-related modifications of cell/nucleus morphology, definitely assigning to Vav1 a crucial role in regulating phenotypical maturation of APL-derived cells (Bertagnolo et al, 2005(Bertagnolo et al, , 2008.…”