Vav1 in differentiation of tumoral promyelocytes

Cellular Signalling 24 (2012) 612–620

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Review

Vav1 in differentiation of tumoral promyelocytes Valeria Bertagnolo ⁎, Federica Brugnoli, Silvia Grassilli, Ervin Nika, Silvano Capitani Signal Transduction Unit, Section of Human Anatomy, Department of Morphology and Embryology, University of Ferrara, Via Fossato di Mortara, 66, 44121, Italy

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Article history: Received 28 October 2011 Accepted 8 November 2011 Available online 28 November 2011 Keywords: Vav1 APL-derived cells Myeloid differentiation Cell nucleus Actin cytoskeleton mRNA processing

a b s t r a c t The multidomain protein Vav1, in addition to promote the acquisition of maturation related properties by normal hematopoietic cells, is a key player in the ATRA- and PMA-induced completion of the differentiation program of tumoral myeloid precursors derived from APL. This review is focussed on the role of Vav1 in differentiating promyelocytes, as part of interconnected networks of functionally related proteins ended to regulate different aspects of myeloid maturation. The role of Vav1 in determining actin cytoskeleton reorganization alternative to the best known function as a GEF for small G proteins is discussed, as well as the binding of Vav1 with cytoplasmic and nuclear signaling molecules which provides a new perspective in the modulation of nuclear architecture and activity. In particular, new hints are provided on the ability of Vav1 to determine the nuclear amount of proteins implicated in modulating mRNA production and stability and in regulating the ATRA-dependent protein expression also by direct interaction with transcription factors known to drive the ATRA-induced maturation of myeloid cells. The reviewed findings summarize the major advances in the understanding of additional, non conventional functions connected with the vast interactive potential of Vav1. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . Vav1 and netrophil-like phenotypical maturation . . . . 2.1. Tyrosine phosphorylation of Vav1 . . . . . . . . 2.1.1. Participation of Vav1 to protein complexes 2.1.2. Phosphorylation of Vav1 on Tyr745 . . . 2.2. GEF-independent activity of phosphorylated Vav1 . 2.2.1. Regulation of actin cytoskeleton . . . . . 2.2.2. Regulation of gene expression . . . . . . 2.3. Vav1 and protein expression . . . . . . . . . . . 2.3.1. Regulation of whole cell proteoma . . . . 2.3.2. Regulation of inner nuclear proteoma . . 3. Vav1 and monocytic/macrophagic differentiation . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: GEF, guanosine exchange factor; APL, acute promyelocytic leukemia; ATRA, All-trans retinoic acid; LIC, leukemia initiating cells; PMA, phorbol 12-myristate 13acetate; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4, 5-bisphosphate; PIP3, phosphatidylinositol 3, 4, 5-trisphosphate; PAK, p21-activated serine-threonine kinase; TMSB10, thymosin beta-10; NFAT, nuclear factor of activated T-cells; AP-1, activator protein-1; NF-κB, Nuclear Factor κB; Sfrs3, Splicing factor, arg/ser rich 3. ⁎ Corresponding author at: Signal Transduction Unit/Laboratory of Cell Biology, Section of Human Anatomy, Department of Morphology and Embryology, Via Fossato di Mortara, 70, Piazzale Eliporto c/o CUBO, 44121 Ferrara, Italy. Tel.: + 39 0532 455571; fax: + 39 0532 207351. E-mail address: [email protected] (V. Bertagnolo). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.11.017

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neutrophils [16,17]. ATRA administration is able to induce at the cellular level the release of the PML–RARα transcriptional repression. The PML–RARα fusion protein, on the other hand, undergoes degradation upon ATRA tratment, an event that, in addition to promote maturation/apoptosis of tumoral promyelocytes (Fig. 1), is at the basis of the eradication of LIC. However, the sole treatment with ATRA does not iduce the complete remission of APL patients, since elimination of LIC requires combined treatment with Arsenic or with conventional chemotherapic agents [18]. This supports the contention that differentiation of tumoral cells and elimination of LIC are uncoupled key events cooperating in the benefit to APL patients. In addition, treatment of human myeloid leukemia cell lines with ATRA and PMA results in changes of their sensitivity to chemotherapeutic drugs, further indicating that advantages in the cure of hematopoietic malignancies may be obtained by combining differentiating agents and conventional anticancer drugs [19–21]. Myeloid differentiation includes characteristic changes of cell morphology which occur in a coordinated sequence leading to acquisition of a highly typical mature phenotype. These architectural changes occur dramatically in the nucleus, making indeed the modifications of the nuclear shape, in addition to the expression of specific pattern of surface antigens, an easy-to-follow marker of neutrophil maturation [22,23]. For the study of neutrophil-like differentiation induced by ATRA, two APL-derived cell lines are universally employed, HL-60 and NB4, which constitute reference cell models since they are blocked at different stages of granulocytic differentiation and reach different levels of neutrophil maturation [24,25]. APL-derived promyelocytes contain levels of Vav1 variably lower than those found in mature neutrophils. Treatments with differentiating doses of ATRA induce a significant increase of Vav1 expression in both cytoplasm and nuclear compartment of primary blasts from APL patients and APL-derived cell lines [26]. The issue of whether the increase of Vav1 observed in differentiation of tumoral promyelocytes is merely designed to the function of the protein in mature cells or, more intriguingly, it is functionally relevant to the maturation mechanism, has been addressed by studies in which the expression of Vav1 was forcedly modulated in HL-60 and NB4 cells. These experiments unequivocally demonstrate that Vav1 is not dispensable for the progression of tumoral promyelocytes along the granulocytic lineage and supports the role of ATRA in regulating the

1. Introduction Vav1 is the sole member of the Vav family of proteins physiologically expressed only in haematopoietic cells, where it acts as an important signal transducer in maturation and immune response of both lymphoid and myeloid cells [1–5]. The role of Vav1 is mainly related to the dynamic regulation of actin cytoskeleton, critical to numerous physical cellular processes of mature hematopoietic cells, including adhesion, migration and phagocytosis [6–10]. In addition, Vav1 activity is specifically required for SDF1α-dependent perivascular homing and subsequent engraftment of hematopoietic stem cells [11]. In both myeloid and lymphoid cells, the best studied function of Vav proteins relies on the tyrosine phosphorylation-dependent activity as GEF for small G proteins [12]. Nevertheless, some functions of Vav1 are independent of its GEF activity and are related to its ability to interact with a number of signaling molecules, in both cytoplasm and nuclear compartment. Inside the nucleus, Vav1 plays its most intriguing role by interacting with components of the DNA-dependent protein kinase complex and with hnRNP proteins as part of transcriptionally active complexes [13–15]. In addition to regulate the acquisition of a mature phenotype by normal hematopoietic cells, Vav1 promotes the agonist-induced completion of the differentiation program of tumoral myeloid precursors. In this context, Vav1 can be regarded as an adaptor potentially involved, in normal and neoplastic cells, in the mechanisms leading the cell to acquire mature properties and/or to reach critical developmental steps, including the rescue from an aberrant differentiative blockade. The current review will focus on the multiple roles played by Vav1 in cell lines derived from APL, as part of interlocked networks of functionally related proteins ended to regulate different aspects of maturation along the neutrophilic and the monocytic/macrophagic lineages. 2. Vav1 and netrophil-like phenotypical maturation ATRA-based therapy represents, until today, the standard cure of APL patients and ATRA treatment of APL constitutes, at present, the only example of successful differentiation therapy of a human cancer, in which tumor cells are induced to complete their maturation to

Cell surface receptors

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Intracellular signaling

ATRA

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Co-repressors Co

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ATRA RXR PML/RARα PML/RAR α

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Fig. 1. Schematic representation of the main mechanisms by which ATRA induces the overcoming of the maturation blockade in APL-derived cells. ATRA acts through nuclear receptors (RARs and RXRs) which are transcription factors for a number of genes encoding signaling proteins that in turn are responsible for the activation of integrated transduction networks. In APL-derived cells, ATRA treatment removes the transcriptional repression due to the PML/RARα protein, derived from the reciprocal and balanced translocation t(15;17) present in a vast majority of APL patients. ATRA also induces the degradation of the chimerical protein, an event that, in addition to restore the role of PML, seems to be at the basis of the eradication of leukemic initiating cells (LIC).

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The tyrosine phosphorylation level of Vav1 increases after ATRA treatment of HL-60 and NB4 cells, opening the question of which kinase/s is/are involved in this process. While in the whole cell the rise of the phosphosphorylation level is almost proportional to the increase in total Vav1 amount, the accumulation of tyrosinephosphorylated Vav1 inside the nuclear compartment seems to be a distinctive feature of the differentiation process, reaching a maximum in ATRA-treated NB4 cells, which achieve more advanced levels of neutrophil maturation [26,28]. In both myeloid and lymphoid cells, Vav1 may be the substrate for receptors with intrinsic tyrosine kinase activity or for membrane and/ or cytoplasmic tyrosine kinases of the Syk/Zap70, Src and Jak families [12]. More recently, the c-Abl kinase has been reported to be specifically involved in regulating the activity of Vav1 in integrin-mediated neutrophil adhesion [29]. Tyrosine phosphorylation of Vav1 depends on its ability to interact with a number of signaling proteins by means of various domains. The interaction between the SH2 domain of Vav1 and phosphorylated proteins is known to recruit activated kinases, which in turn can phosphorylate Vav1 [12]. The Syk/ZAP-70 tyrosine kinases contain two SH2 domains, a tandem sequence that might confer high specificity in tyrosine kinase- mediated signaling. In addition, both Syk and ZAP-70 contain a consensus binding sequence for the Vav1 SH2 domain that seems to be critical for antigen receptor-mediated signal transduction in hematopoietic cells [30]. Activation of Syk occurs in mature neutrophils, in which it regulates migration [31] and the formation of lamellipodia during phagocytosis [32]. Syk is also activated in HL-60 cells treated with ATRA [33,34] in which tyrosine phosphorylated Syk associates with the Vav1-SH2 domain. The formation of Vav1/Syk complexes also occurs inside the nuclear compartment and strongly increases during the differentiation process [34], suggesting a compartmentalized role for Syk in this cell model. The role of Syk in phosphorylating Vav1 has been demonstrated in HL-60 cells by means of in vitro assays and confirmed by the use of a pharmacological model of Syk inhibition, in which both HL-60 and NB4 cells were treated with Piceatannol [34,27], a tyrosine kinase inhibitor with a reported selectivity for Syk [35,36]. The Syk-dependent tyrosine phosphorylation of Vav1 is not relevant for the expression of the surface marker CD11b during the ATRA-induced phenotypical differentiation of both HL-60 and NB4 but rather plays a crucial role in regulating the reorganization of cell architecture. In fact, when Syk activity is down-modulated, the ATRA-induced 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 [27]. The inability of Piceatannol to abrogate completely the ATRA-induced tyrosine phosphorylation of Vav1 in both HL-60 and NB4 cells [27] indicates that other kinase/s, in addition to Syk, are recruited by ATRA in these cell models. The use of PP1 and AG490, inhibitors of Src and Jak tyrosine kinase families, respectively, failed to affect to any significant extent the tyrosine phosphorylation of Vav1 [23], leaving open the question of which tyrosine kinases phosphorylate Vav1 during maturation of tumoral promyelocytes.

Vav1 as interacting motifs [12]. In this context, SLP-76, an adaptor protein substrate for ZAP-70 and Syk tyrosine kinases, has been reported to associate, via tyrosine-phosphorylated residues in its NH2-terminal domain, with the SH2 domain of Vav1, after ligation of the T-cell antigen receptor [37,38]. SLP-76 is expressed in multiple hematopoietic lineages including T cells, platelets, and neutrophils. SLP-76-mediated signaling is dependent on its multiple protein interaction domains and the signaling molecules, including Vav1, used upstream or downstream of SLP-76 are similar across cell types [39]. The adaptor protein SLP–76 has been identified as a phosphorylated protein interacting with the SH3–SH2–SH3 fragment of Vav1 in differentiating HL-60 cells. Vav1-associated SLP76 is more abundant in nuclei than in whole-cell lysates, indicating a preferential relationship of these two molecules inside the nuclear compartment [34]. Some of the Vav1-interacting molecules play a role in downmodulating Vav1 tyrosine phosphorylation. A potential negative regulator of Vav1 is Cbl, which is known to down-modulate Syk/ZAP-70 and other tyrosine kinases potentially responsible of Vav1 phosphorylation [40]. A direct interaction of Cbl with Vav1 has also been demonstrated, requiring the whole SH3–SH2–SH3 COOH-terminal domain of Vav1 and a proline-rich sequence of Cbl [41]. Cbl interacts with Vav1 in HL-60 cells only in the cytoplasm and is strongly phosphorylated in response to ATRA treatment. Even though the Vav1/Cbl interaction in HL-60 cells occurs also in control conditions and requires the entire SH3–SH2–SH3 domain of Vav1, the precise role of Cbl in regulating Vav1 in this cell model has not been addressed. On the other hand, exclusive of the ATRA treatment of HL-60 seems to be the compartmentalized association between Vav1 and interacting proteins during ATRA treatment, since Cbl/ Vav1 complexes are located in the cytoplasm while SLP-76/Vav1 complexes reside in the inner nuclear compartment [34]. In differentiating APL-derived cells Vav1 seems to be recruited by one or more signal transduction cascades directed to the nucleus and involving Cbl and SLP-76, which may then discretely regulate the amount of Vav1 in the cytoplasmic and nuclear compartments (Fig. 2). The interaction of Vav1 with both Cbl and SLP-76 in HL-60 cells may be correlated to the transmembrane signaling mediated by CD38, an early biomarker of ATRA-induced differentiation in this cell line [42]. Very recently it has been demonstrated that the expression of a cytosolic deletion mutant of CD38 fails to up-regulate ATRAinduced proteins such as CD11b, Vav1 and Fgr, this latter able to phosphorylate Vav1 after ATRA treatment of HL-60 cells [43]. The Vav1-associated protein complexes identified in HL-60 cells also contain the tyrosine kinase Syk, being Vav1/Cbl/Syk complexes present only in cytoplasm and Vav1/SLP-76/Syk complexes only inside the nuclear compartment [34]. Since these associations are partially present in control conditions and strongly increase after ATRA treatment, during the maturation of APL-derived myeloid precursors, a sequence of signals originated from membrane receptors and directed to the nuclear compartment seems to regulate the amount of tyrosine phosphorylated Vav1 inside the nucleus (Fig. 2). In both cells and nuclei of HL-60 cells, other signaling molecules associate with phosphorylated Vav1 as a consequence of ATRA treatment. They include the γ1 isoform of PI-PLC and the p85 regulatory subunit of PI3K [28]. In this context, Vav1, due to its nuclear localization sequence, may have a specific role in driving inside the nuclear compartment signaling molecules that, independently or in collaboration with Vav1, will exert their role in this cell compartment.

2.1.1. Participation of Vav1 to protein complexes with signaling molecules The optimal phosphorylation of Vav1 requires the association with adaptor molecules that facilitate the spatial proximity between Vav1 and the upstream tyrosine kinases. These associations often require the engagement of either the SH3 or the SH2 domains of

2.1.2. Phosphorylation of Vav1 on Tyr745 Tyrosine phosphorylation of Vav1 was originally investigated almost exclusively in relation to the function of Vav1 as a GEF. A crucial role in this context is played by Tyr174, even if other mechanisms have emerged in the last few years as regulators of Vav1 GEF activity, and Tyr174 is conversely involved in roles of Vav1 not mediated by

maturation process, in terms of both surface antigens expression and modifications of cell/nucleus morphology [26,27]. 2.1. Tyrosine phosphorylation of Vav1

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Cytokines (CD38?)

Cbl SLP-76

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Vav1 P

Vav1

Vav1 PI3K actin PIP2

SLP-76

Vav1

PIP2

Syk P Vav1

P

Vav1 PI3K actin

PIP2

PIP3

Fig. 2. Syk-dependent tyrosine phosphorylated Vav1 in the regulation of cell/nucleus architecture of differentiating HL-60 cells. ATRA treatment induces the activation of the tyrosine kinase Syk and its recruitment in compartmentalized complexes with the adaptor proteins Cbl and SLP-76, ended to facilitate the tyrosine phosphorylation of Vav1 by Syk. In both cytoplasmic and nuclear compartments, Vav1 tyrosine phosphorylated by Syk regulates PI3-K activity by modulating its interaction with the actin associated phosphoinositide pools. A detailed description of this pathway is included in the text.

GEF activity [4]. In addition to Tyr174, also Tyr142 and Tyr160 are phosphorylated in activated Vav1. It has also been suggested that phosphorylation of tyrosines located inside the acidic region of Vav1 may allow Tyr142, Tyr160, and Tyr174 to become docking sites for kinases which can then phosphorylate additional tyrosine residues [44,45]. Recently, several of the tyrosine residues at the carboxyl terminus of Vav1 have been shown to be phosphorylated in cancer cells, raising the possibility that many, if not all, of the 31 tyrosine residues of Vav1 may play important roles in Vav1 function [46]. Members of the Syk/Zap70 and Src families of tyrosine kinases phosphorylate the residue Y174 of Vav1 in mature neutrophils, mediating in this way neutrophil migration in vitro and neutrophil recruitment during the inflammatory response in vivo [31]. However, the issue of which kinases are responsible of phosphorylating the other Vav1 tyrosine residues has not been addressed as yet in this cell model. In APL-derived cells, ATRA induces the phosphorylation of Tyr174 in NB4 but not in HL-60 cells. In any case, this event is independent of the induced activation of Syk and is not relevant for the activity of ATRA in this cell line [47,48]. This suggests that in tumoral myeloid precursors, the ATRA-induced phosphorylation of Tyr174 occurs in parallel with differentiation and may constitute a marker of the acquisition of a mature phenotype. This is confirmed by the failure of ATRA to induce the phosphorylation of Tyr174 in HL-60 cells, that reach indeed only a partially differentiatiated phenotype when compared to NB4 cells [48]. The search for tyrosine residues, other than Tyr174, phosphorylated as a consequence of ATRA treament, performed by a proteomic approach on NB4, allowed to identify Tyr745, located within a highly conserved Vav1 sequence. This residue is crucial in supporting the ATRAinduced acquisition of maturation-related features, in terms of CD11b expression and migratory capability of NB4 cells [47]. Even if Tyr745 has never been correlated with the known roles of Vav1, multiple

sequence alignment analysis of proteins from different species indicates that this is a highly conserved aminoacid, likely involved in physiological, although unknown, roles of Vav1. Inhibition studies have ruled out any role of Syk in phosphorylating Tyr745 as a consequence of ATRA treatment [47] and, at present, no data are available about the involved tyrosine kinase. Finally, since Tyr745 is located inside a short helix on the SH2 domain of Vav1, its phosphorylation could be an event secondary to phosphorylation of other tyrosine residues, which may induce conformational changes of Vav1 allowing Tyr745 to become accessible to a specific tyrosine kinase. 2.2. GEF-independent activity of phosphorylated Vav1 GEF activity of Vav1 has long been regarded as the key for transferring the signal from activated receptors to the cytoskeleton, in which actin seems to be a preferred target of the Vav1-dependent GEF activity [49]. Vav1 is a preferential exchange factor for Rac1, which in turn may activate PIP 5-kinase that phosphorylates PIP to PIP2, an activator of actin-binding proteins, like talin and vinculin, involved in the attachment of the cytoskeleton to the cell membrane. Another potential target for the GEF activity of Vav1, Cdc42, may regulate actin polymerization through the activation of the WASP protein [49]. Finally, small G proteins activated by Vav1 play an essential role in regulating actin cytoskeleton dynamics by also interacting with the PAK family of actin-regulatory enzymes [50]. ATRA treatment induces an increase of the total GEF activity in HL60 cells independent from Vav1 [34], indicating that, in this cell model, the Syk-dependent tyrosine phosphorylation of Vav1 is not ended to regulate its GEF activity and that alternative pathways have to be considered to explain the mechanism by which Vav1 affects the organization of cytoskeleton and nucleoskeleton during maturation of tumoral promyelocytes.

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2.2.1. Regulation of actin cytoskeleton In both lymphoid and myeloid cells, like other proteins with a GEF activity, Vav1 mediates a number of cytoskeletal-associated cellular processes, including the formation of immunological synapse and phagocytosis of T cells (reviewed in 49) and chemotaxis of neutrophils and macrophages [51–53], all requiring profound modification of actin organization. On the other hand, Vav1 may mediate actin reorganization through other, GEF-independent mechanisms, based on the presence in its structure of a number of tyrosines and domains potentially involved in protein–protein interactions. A role of Vav1 in actin polymerization as an adapter protein that links signaling and cytoskeletal molecules has been described in T cells, in which Vav1 binds constitutively Talin and Vinculin, anchoring the actin cytoskeleton to the plasma membrane. Also the interaction of Vav1 with the cytoskeletal protein Zyxin and Dynamin 2 has been demonstrated in the same cell model [49,54]. In ATRA-treated HL-60 cells, the mechanism by means of which tyrosine-phosphorylated Vav1 regulates actin cytoskeleton implies the interaction of Vav1 with the p85 regulatory subunit of PI3K. Studies aimed to establish the functional significance of this interaction have demonstrated that, in maturating myeloid precursors, PI3K activity closely depends on its association with Vav1 tyrosine phosphorylated by Syk and that when Vav1/PI3K interaction and/or PI3K activity are abrogated, the phenotypic differentiation of ATRA-treated HL-60, in terms of both surface antigen expression and modifications of cellular/nuclear morphology, is compromised [55,23]. Also actin is present in the ATRA-induced protein complexes containing Vav1 and PI3K in HL-60 cells. Remarkably, when the Vav1/PI3K association is impaired by down-modulation of Syk activity, the formation of PI3K/actin complexes is reduced [23], suggesting that the interaction of PI3K with tyrosine-phosphorylated Vav1 is essential for its association with actin. Since the recovery of 3-phosphoinositides is strongly reduced when the Vav1dependent interaction between PI3K and actin is abrogated, it can be concluded that Vav1 regulates the physical contact of PI3K with its cytoskeleton-associated substrates (Fig. 2). These evidences assign to Vav1/PI3K interaction a prominent role in the regulation of cytoskeleton, alternative to the described function of 3phosphoinositides in modulating GEF activity of Vav1 [56] and indicate that, in addition to play a regulatory role in Vav1 activation, PI3K activity may itself be regulated by Vav1. PI3K is likely to play essential roles in granulocytic differentiation of tumoral myeloid precursors, considering that both downmodulation of its expression and pharmacological inhibition of its activity during ATRA treatment significantly reduce the tendency of HL-60 cells to acquire the differentiated phenotype [55]. The downstream effects of PI3K activity observed during the induced neutrophil-like differentiation support the notion that PI3K is recruited in the path controling cytoskeleton in mature granulocytes. In fact, PI3K is activated in response to chemotactic factors in both murine and human neutrophils [57–59] in which newly produced PIP3 is involved in determining the localization and possibly the crosslinking/stabilization of actin filaments [60–62]. In vitro experiments have demonstrated that PI3K may directly affect actin-related modifications of cytoskeleton by modulating PAK kinase activity [63]. At any rate, PI3K has probably a more direct influence on cytoskeleton by determining the amount of the inositol-containing lipids, that have emerged as major players in regulating actin assembly at several levels and with different mechanisms, including the direct interaction with other cytoskeletal proteins, such as vinculin and gelsolin [64,65]. Since, in ATRA-treated promyelocytes, both PI3K activity and the modifications of the cell/nucleus architecture depend on the formation of Vav1/PI3K complexes, Vav1 may be important for targeting PI3K to its substrates, particularly inside the nucleus. The association of Vav1 with other lipid modifying enzymes, including specific PI-PLC

isoforms [28], further supports its role in determining the composition of the actin-associated phosphoinositide pool and, ultimately, in regulating actin polymerization in differentiating HL-60 cells. 2.2.2. Regulation of gene expression As a consequence of ATRA administration, Vav1 tyrosinephosphorylated by Syk accumulates inside the nuclear compartment of APL-derived cells and becomes involved in the reorganization of their nuclear architecture. These structural changes are at the basis of both transcription and post-transcriptional events, suggesting indeed that Vav1 may play a prominent role also in regulating ATRArelated gene expression. An array analysis performed on HL-60 cells and focussed on genes coding for cytokine and cytokine receptors, in turn involved in the differentiative program of tumoral promyelocytes [66,67], indicates that the inhibition of the Syk-dependent tyrosine-phosphorylation of Vav1 during ATRA treatment prevents the ATRA-induced gene expression [26]. In particular, tyrosine-phosphorylated Vav1 is essential for the expression of TMSB10, a small G-actin binding protein that induces depolymerization of the intracellular F-actin pools and thus deeply affects actin architecture [68,69], further correlating Vav1 with actin cytoskeleton. Vav1 tyrosine-phosphorylated by Syk is also involved in regulating the ATRA-induced expression of the Notch homolog, that mediates cell fate decisions during hematopoiesis [70] and whose signaling might be necessary for the proliferation and survival of AML cells, possibly regulating the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein [71]. The silencing of Vav1 expression during ATRA admistration has unambiguously allowed to ascertain its role in regulating ATRA-dependent gene expression, further confirming that the up-regulation of Vav1 is not only a maturation-related phenomenon but is also a key event in granulocytic differentiation of tumoral myeloid precursors. In myeloid and lymphoid cells, Vav1 seems to be involved in regulating DNA transcription by direct interaction with, or as a facilitator of, transcription factors. In particular, Vav1 regulates NFAT, AP-1 and NFκB in T-cells in response to TCR stimulation, and exerts a specific role in regulating the CREB-dependent gene transcription [72,73]. Direct evidence for the presence of Vav1 in active transcriptional complexes with NFAT and NF-kB-like has been demonstrated in mast cells, in which Vav1 acts as a facilitator of transcriptional activity [15]. In APL-derived cells, nuclear Vav1 associates with PU.1 [74], a transcription factor induced by ATRA and with a crucial role in the completion of granulocytic differentiation of tumoral myeloid precursors [75]. On the other hand, in NB4 cells, like in other tumoral myeloid precursors [76], PU.1 regulates the expression of Vav1 induced by ATRA [74]. In tumoral myeloid precursors, PU.l is a major determinant of the myeloid expression of CD11b [77,78], an integrin receptor whose surface expression increases during differentiation of APL-derived cell lines [79]. The over-expression of PU.1 might influence phenotype and restore differentiation of primary myeloid leukemic blasts [80], and its silencing counteracts the ATRA ability to induce the expression of the granulocytic marker CD11b [75]. In NB4 cells treated with ATRA, PU.1 is recruited to its consensus sequence within the CD11b promoter [74] and may be used by ATRA to promote CD11b expression during the late stages of the maturation of APL-derived cells. Although unphosphorylated Vav1 is also recruited to the PU.1 consensus sequence on the CD11b promoter in untreated NB4 cells, the participation of Vav1 to molecular complexes including PU.1 has been ruled out. ATRA treatment, by inducing an increase in Sykdependent tyrosine phosphorylation of Vav1, displaces Vav1 from existing molecular complexes on the CD11b promoter [74], an event that seems to promote the formation of PU.1-containing complexes. In fact, when the amount of Vav1 is forcedly reduced or its tyrosine phosphorylation is inhibited during the differentiation treatment,

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role in tumorigenesis and in the adverse evolution of the disease [87,88], Vav1 may promote the differentiation of tumoral promyelocytes by also targeting metabolic pathways. Similarly, the proteasome component “splice isoform 2 subunit α3” is down-modulated as a consequence of reduced Vav1 expression in both HL-60 and NB4 cells [27]. Since proteasome is the major cellular proteolytic machinery [89], Vav1 may also be involved in regulating protein degradation during ATRA dependent maturation of tumoral promyelocytes. Also the expression of the microtubule component α-tubulin is affected by the down-modulation of Vav1 during ATRA treatment [27], indicating that Vav1, in addition to participate to the assembly/ reorganization of cytoskeleton, is involved in the regulation of the expression of cytoskeleton components.

the formation of a PU.1-containing complex is compromised [74]. It is then conceivable that Vav1, and in particular Vav1 tyrosinephosphorylated by Syk, regulates the recruitment of PU.1 to its consensus sequence on the CD11b promoter region and, possibly, the expression of this surface antigen (Fig. 3). 2.3. Vav1 and protein expression Recent studies have used the proteomic approach to evaluate protein expression during differentiation/apoptosis induced by different agonists in APL-derived cells, demonstrating that ATRA modulates the expression level of structural and signal transduction proteins as well as of molecules involved in the different phases of protein synthesis [81–84].

2.3.2. Regulation of inner nuclear proteoma The nucleus of tumoral myeloid precursors differentiating along the neutrophilic lineage is a crucial cell compartment since ATRA acts through nuclear receptors (RARs) that are ligand-regulated transcription factors [24,25,90]. The activation of RARα-mediated gene transcription [91] and the regulation of posttranscriptional events [83] result in profound modulations of the nuclear protein pools and are accompanied by fundamental changes in nuclear architecture and activity. Deep changes of the protein pool also occur in the inner nuclear compartment of NB4 cells in response to ATRA [92]. The majority of up-regulated proteins are either part of the transcription machinery or variably involved in RNA processing, in agreement with the role played by ATRA nuclear receptors in regulating gene transcription. Since their total mRNA level is not affected, it is conceivable that ATRA only triggers their transport inside the nuclear compartment of differentiating NB4 cells. On the other hand, for the majority of the identified proteins whose nuclear amount was downmodulated by ATRA, also a decrease in their total mRNA was demonstrated. Experiments in which the expression of Vav1 is down-modulated during ATRA treatment of NB4 cells demonstrate that Vav1 regulates the nuclear amount and/or the mRNA levels of ATRA-modulated proteins [92]. In particular, the response to ATRA involves, inside the

2.3.1. Regulation of whole cell proteoma In HL-60 and NB4 cells, Vav1 behaves as a key tool recruited by ATRA to regulate protein expression. In fact, down-modulation of Vav1 during ATRA administration affects the agonist-dependent expression of proteins associated to cytoskeleton, involved in proliferation/ apoptosis, as well as of molecules implicated in metabolism, synthesis, folding and degradation of proteins [27]. The majority of the so far identified proteins are affected by Vav1 only in one of the two 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 [79]. Exclusive of NB4 cells is the Vav1-related expression of the Sfrs3 member of SR proteins, known as nonsnRNP splicing factors that may affect both constitutive and alternative splicing of mRNA [85]. Proteins whose expression is affected by Vav1 in both HL-60 and NB4 cells may constitute a common part of the signaling activated by ATRA in APL-derived promyelocytes. They include 14-3-3ε, specifically involved in the caspase networks [86], suggesting that Vav1 may be critical in determining the mechanism of caspase activation in APL. Vav1 also affects the ATRA-dependent expression of α-enolase, a multifunction protein involved in glycolysis. Since α-enolase is up-regulated in the sera of a number of cancer patients, in which it seems to have a

Cell surface receptors

CD11b

Signal transduction pathways

Syk Vav1 SRs hnRNPs

Vav1

Vav1

P

CD11b

Vav1

P

Vav1

Vav1 SRs hnRNPs

PU.1

PU.1

CD11b

Gene transcription Fig. 3. Schematic representation of Vav1 as a molecule able to regulate different aspects of ATRA-dependent gene expression. In APL-derived cells, Vav1 is involved in regulating the ATRA-dependent gene transcription, in terms of both mRNA and protein expression. In particular, Vav1 tyrosine-phosphorylated by Syk is part of molecular complexes with the transcription factor PU.1 and may be responsible of regulating the recruitment of PU.1 to its consensus sequence on the CD11b promoter. In addition, Vav1 may participate to RNA processing by carrying into the nucleus molecules involved in modulating mRNA production and stability, like SR and hnRNPs proteins.

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nuclear compartment, a Vav1-dependent modulation of hnRNP D, hnRNP K and hnRNP H3. hnRNPs are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism [93] and variously implicated in cancer development [94]. Since some hnRNP proteins continuously shuttle between the nucleus and the cytoplasm [95,96], Vav1 may act as a scaffold protein specifically involved in regulating the redistribution between cytoplasm and nuclear compartment of proteins with specific roles in RNA processing (Fig. 3). Also the ATRA-dependent nuclear amount of the splicing factor Sfrs9 member of the highly conserved SR proteins depends on Vav1 [93]. SR proteins are non-snRNP splicing factors that may affect both constitutive and alternative splicing of mRNA [85] and crucial regulators of mRNA export in different cell models [97–99]. Since also the nuclear amount of HNRPDL and of C14orf166 seems to be regulated by Vav1, it can be proposed that Vav1 has a prominent role in protein shuttling between the nucleus and the cytoplasm. In differentiating NB4 cells, Vav1 affects the ATRA-dependent gene transcription in terms of mRNA levels [92]. This is in agreement with the finding that the proteins whose ATRA-dependent nuclear amount is regulated by Vav1 are almost all involved in mRNA processing and that Vav1 is present in transcriptional complexes of ATRA-treated NB4 cells [74]. In addition, Vav1 has a role in determining both mRNA levels and nuclear amount of lamin-B1, a key regulator of nuclear functions. A lamin B1-containing nucleoskeleton is required to maintain DNA and RNA synthesis and ongoing synthesis is fundamental for global nuclear architecture in mammalian cells [100]. In tumoral promyelocytes, a decrease in the nuclear amount of lamin-B1 as a consequence of ATRA administration has been described, consistent with the reduced amount of lamin-B1 observed in the nuclear envelope of mature neutrophils from peripheral blood [101]. This occurrence, whose significance remains to be fully elucidated, may be relevant to reduce mechanical resistance of the cell, in order to facilitate migration through tight tissue spaces, or to take part to an apoptosis-promoting mechanism involved in death of post-mitotic mature granulocytes. Finally, in NB4 treated cells, Vav1 modulates the nuclear amount of BCAS2, a small nuclear protein recently identified as negative regulator of p53 and suggested as a molecular target for cancer therapy [102]. The Vav1-dependent down-regulation of BCAS2 induced by ATRA in NB4 cells indicates that also in promyelocytes this protein correlates with tumoral properties and corroborates the likelihood that Vav1 is a potential intermediate target for therapies aimed to down-modulate BCAS2. 3. Vav1 and monocytic/macrophagic differentiation HL-60 and NB4 cell lines can be differentiated, with dynamics not identical to that regulating their neutrophil-like maturation, to monocytes/macrophages by PMA, a stable analogue of 2, 3diacyloglycerol [19,103,104]. Both expression and tyrosine phosphorylation of Vav1 increase during the PMA-induced acquisition of a monocyte-like phenotype of HL-60 and NB4 cells [48], consistently with the notion that mature monocytes express Vav1 and that proper amounts of the protein are necessary for their inflammation related functions [7,105]. On the other hand, and in constrast to what observed in the ATRA-treatment of the same cell line, Syk is not recruited by PMA [48], consistent with the notion that, at least in HL-60 cells, Syk might exert a narrower role, restricted to directing cells toward granulocyte differentiation [33]. In both HL-60 and NB4 cells, PMA induces a relevant increase of Vav1 phosphorylation on Tyr174 [48], even if a role as a GEF was not demonstrated for Vav1 in this model of cell differentiation. A clear correlation between Syk and tyrosine phosphorylation of Vav1 was demonstrated in HL-60 differentiated with PMA, in which the kinase regulates both actin dynamics and the Vav1-RhoA activation pathway, ended to control the roles played by mature cells in immune response, including complement-mediated phagocytosis [32].

By silencing the expression of Vav1 induced by PMA, a crucial role for Vav1 has been demonstrated also in differentiation of APL-derived cells to monocytes/macrophages [48]. In particular, Vav1 regulates the expression of the CD11b surface antigen, which is induced by PMA and constitutes a marker for monocyte differentiation. Also cell adhesion is affected by down-modulation of Vav1 during PMA treatment of HL-60 and NB4 cells, in agreement with the data obtained on macrophages from Vav1 _/ _ mice, which show a smaller adhesive area or a decreased adhesion efficiency [6]. In PMA-treated NB4 cells, contrarily to what observed during granulocytic differentiation, Vav1 down-modulation does not affect expression and architectural organization of α-tubulin [48], indicating that, during the maturation process of APL-derived cells, Vav1 exerts an agonist- and lineage-specific role in regulating this microtubule component. Concerning microtubule organization, since changes in microtubule dynamics contribute to the reduced migration speed of Vav1_/_ macrophages in response to CSF-1 [6], it can be speculated that the role of Vav1 is restricted to the control of the motility of mature cells. In HL-60 and NB4 cells treated with PMA, Vav1 regulates actin expression [48], further confirming that Vav1, besides to be involved in the formation of filaments, takes part to cytoskeleton reorganization also as a modulator of protein expression. The modifications of cell shape in the different cell processes seem to be regulated by the existence of a thin, membrane-bound F-actin network called F-actin cortex [10]. A wide variety of contractile Factin networks with different architectures and polarity have also been found near cell adhesion surfaces, correlated with the migratory capability of adherent cells [10]. Furthermore, the shape of the nucleus of adherent cells is tightly regulated through a perinuclear actin cap, which is located above and around the interphase nucleus [106]. Defective actin-cap formation has been found in lymphocytes from Vav-deficient mice, clearly correlating Vav1 activity with the regulation of cell shape [107], and, in macrophages, Vav proteins contribute to the maintenance of normal morphology and migratory behavior [105]. The existence of an agonist-induced F-actin network, in which F-actin colocalyzes with Vav1 has been demonstrated in PMA-treated adherent NB4 cells [48]. Since cytoplasmatic processes in PMA-treated adherent cells are indicative of migratory activity [10], Vav1/F-actin co-localization in cytoplasm protrusions is suggestive of a synergy of the two molecules in controling cell motility. The strong Vav1/F-actin co-localization observed at the nuclear periphery and above the nucleus suggests that the two proteins may cooperate in regulating the shape of the nucleus through an actin filament structure similar to the perinuclear actin cap originally described by Khatau et al. [106]. 4. Conclusion The present review focuses on the role of the multidomain protein Vav1 in promoting and sustaining the completion of the differentiation program of tumoral promyelocytes, as an essential element of interplaying networks of functionally related proteins engaged to regulate different aspects of myeloid maturation. The majority of the roles played by Vav1 in this cell model are alternative to the best known function of Vav1 as a GEF for small G proteins, reflecting the great interactive and regulatory potential of Vav1, which make the full understanding of its functions a very difficult, yet fascinating issue. The participation of Vav1 to a signaling sequence originated from membrane receptors and directed to the nuclear compartment confers to Vav1 compartimentalized strategic roles in regulating the maturation process of tumoral promyelocytes. Inside the nucleus of APLderived cells, Vav1 seems to play its most intriguing role by regulating the expression of CD11b, a surface marker of both granulocyte and monocyte differentiation, and of a number of ATRA-modulated proteins.

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