Elsevier

Cellular Signalling

Volume 45, May 2018, Pages 132-144
Cellular Signalling

New insights into the Vav1 activation cycle in lymphocytes

https://doi.org/10.1016/j.cellsig.2018.01.026Get rights and content

Highlights

  • The basal inactive state of Vav1 is unexpectedly under the control of PLC γ1 and Slp76 in lymphocytes

  • The kinetics and amplitude of Vav1 phosphorylation are under the control of different kinases in lymphocytes

  • Vav1 phosphorylation does not follow the canonical TCR-Lck-Zap70 cascade

  • Specific compound mutations of Vav1 regulatory phosphosite differentially affect signaling diversification events in T and B lymphocytes

Abstract

Vav1 is a hematopoietic-specific Rho GDP/GTP exchange factor and signaling adaptor. Although these activities are known to be stimulated by direct Vav1 phosphorylation, little information still exists regarding the regulatory layers that influence the overall Vav1 activation cycle. Using a collection of cell models and activation-mimetic Vav1 mutants, we show here that the dephosphorylated state of Vav1 in nonstimulated T cells requires the presence of a noncatalytic, phospholipase Cγ1–Slp76-mediated inhibitory pathway. Upon T cell stimulation, Vav1 becomes rapidly phosphorylated via the engagement of Lck and, to a much lesser extent, other Src family kinases and Zap70. In this process, Lck, Zap70 and the adaptor protein Lat contribute differently to the dynamics and amplitude of the Vav1 phosphorylated pool. Consistent with a multiphosphosite activation mechanism, the optimal stimulation of Vav1 can only be recapitulated by the combination of several activation-mimetic phosphosite mutants. The analysis of these mutants has also unveiled the presence of different Vav1 signaling competent states that are influenced by phosphosites present in the N- and C-terminal domains of the protein.

Introduction

Vav1 mainly works as a tyrosine phosphorylated-regulated Rho guanosine nucleotide exchange factor (GEF), a catalytic activity that allows the rapid transition of Rho GTPases from the inactive (GDP-bound) to the active, GTP-bound state during cell signaling [1,2]. In addition, it displays in some contexts adaptor functions that allow the regulation of downstream signals using catalysis-independent mechanisms [1,3]. For example, Vav1 can promote the Cbl-b-mediated degradation of the intracellular domain of Notch1 [4,5] and the Ca2+-dependent stimulation of the nuclear factor of activated T-cells (NFAT) [[6], [7], [8], [9]], a transcriptional factor essential for the expression of cytokines and other activation-connected proteins in lymphocytes [10]. These adaptor functions can be dependent (NFAT) or independent (Notch1) of the phosphorylation state of Vav1 [1,[3], [4], [5]].

Vav1 is characterized by a multidomain structure that harbors calponin homology (CH), acidic (Ac), Dbl homology (DH), pleckstrin homology (PH), C1-subtype zinc finger (C1), proline-rich (PRR), SH3, and SH2 regions [1] (Fig. 1A). These domains play roles related to the intramolecular regulation of the protein (CH, Ac, PH, SH3) [1,[11], [12], [13], [14]], the activation step (SH2, SH3) [[15], [16], [17]], the catalytic process (DH, PH, C1) [13,14,18,19], the establishment of interactions with protein partners (PRR, SH3, SH2), and the catalysis-independent regulation of NFAT (CH) and Notch1 (the two SH3s) [1,6,8,9,14,[20], [21], [22]]. Whereas the basis of the regulation of the catalytic activity of Vav proteins is well characterized at the structural level [18,19], the mechanism of stimulation of the NFAT route by Vav1 is still poorly understood. However, it is known that it involves the stimulation of phospholipase Cγ (PLCγ) which, upon the IP3-mediated mobilization of Ca2+ from intracellular stores, favors the stimulation of the phosphatase calcineurin by Ca2+-calmodulin [9]. This phosphatase promotes in turn the dephosphorylation of cytoplasmic NFAT and its final shuttling to the nucleus (Fig. 1B). Unlike the case of the catalysis-dependent pathways, the NFAT route requires the parallel action of Vav1 and other TCR-stimulated signaling pathways to achieve full activation in cells [6] (Fig. 1B). As a result, this pathway can be further stimulated by the TCR even when cells express constitutively active Vav1 proteins (e.g., Vav1Δ835–845) [23]. Also in contrast to the catalysis-dependent pathways, the NFAT route cannot be activated by constitutively active Vav1 proteins lacking the CH domain (e.g., Vav1Δ1–66, Vav 1Δ1–144, Vav1Δ1–184) (Fig. 1B) [1,6,8,9,14,20,21].

The inactive state of Vav proteins is maintained by inhibitory intramolecular interactions established by the CH, Ac and most C-terminal SH3 (CSH3) regions with the catalytic DH-PH-C1 core (Fig. 1A) [12,23]. This “closed” structure shifts towards an “open” conformation upon the phosphorylation of tyrosine residues located in the Ac (Y142, Y160, Y174), CSH3 (Y836) and, to a lesser extent the C1 region (Y541, Y544) [21,23] (Figs. 1A and S1). Consistent with this model, mutations that eliminate the foregoing inhibitory structure lead to the generation of phosphorylation-independent, constitutively active proteins [1,13,14,24]. This activation can be unexpectedly achieved using both Tyr to Glu and Tyr to Phe mutations [12,14,21,23], an effect probably due to the implication of the side chains of these phosphosites in the stabilization of the intramolecular inhibitory structure [11,12]. In the case of antigen receptors, the Vav1 activation step requires the stepwise action of adaptors and PTKs that, in many cases, are cell type-specific. For example, in T cells, this step entails the membrane tethering of Vav1 by adaptor molecules (e.g., Grb2, Nck, Lat) and, subsequently, the ensuing phosphorylation by TCR-activated protein tyrosine kinases [1,25].

Despite these advances, there are still open questions associated with the mechanism of activation of Vav1 and rest of family members. For example, it is still unclear which PTK(s) phosphorylate them downstream of antigen receptors, as many kinases (Lck, Fyn, Hck, Syk) are capable of promoting both the phosphorylation and catalytic activation of Vav proteins in vitro [1,2,13,24]. The multiple phosphorylation sites involved in this activation step also pose the possibility that Vav proteins could adopt different conformational and signaling states depending upon the number of tyrosine residues phosphorylated at a given time on the molecule [23]. In line with this, we do not know whether the engagement of the catalytic and adaptor functions of these proteins is subjected to similar signaling constraints. We have addressed those issues in this work using a multifaceted approach based on the utilization of phosphosite-specific Vav1 antibodies, wild-type and mutant cell models, and activation-mimetic Vav1 phosphosite mutants. In addition, we used biological readouts that allowed us to monitor the catalytic and adaptor activities of Vav1 under each of the above experimental conditions.

Section snippets

Results

Lck is the main tyrosine kinase involved in optimal Vav1 activation in Jurkat cells.

We followed two intertwined approaches to assess the contribution of upstream PTKs to Vav1 signaling. On the one hand, we analyzed the phosphorylation status of endogenous Vav1 immunoprecipitated from parental, Zap70-deficient (P116 cell line) and Lck-deficient (J.Cam1.6 cell line) Jurkat cells [26,27] using immunoblots with antibodies to either general (pTyr) or specific Vav1 phosphorylated tyrosine residues (Y

Discussion

Despite the functional connection of Vav family proteins with tyrosine phosphorylation events, this key regulatory step still constitutes a black box from a mechanistic point of view. This problem is particularly acute in lymphocytes, since both the activation and downstream signaling of these proteins can be potentially influenced by a large number of PTKs, coreceptors, phosphatases, and adaptor molecules [1]. Using Vav1 as a model, we have seen here that this regulatory process is even more

Antibodies

The polyclonal antibody to the Vav1 DH domain (Ref. 302-5) was raised in rabbits using a maltose binding protein-Vav1 DH fusion protein previously purified from Escherichia coli according to standard techniques. Polyclonal antibodies to the indicated Vav1 phosphosites have been described before [21,23]. Other polyclonal antibodies used include those to Lck (Santa Cruz Biotechnology), GAPDH (Santa Cruz Biotechnology), PLCγ1 (Cell Signaling Technology), Slp76 (Cell Signaling Technology), p-PKD

Data availability

No datasets have been generated in this work. All reagents generated by our lab for this work are freely available upon request.

Acknowledgements

X.R.B. is supported by grants from the Castilla-León Government (BIO/SA01/15, CSI049U16), Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-64556-R), Worldwide Cancer Research (14-1248), Ramón Areces Foundation, and Spanish Society against Cancer (GC16173472GARC). M.B. and S.R-F. salaries have been supported by a JAE-Predoc (CSIC) and MINECO FPI (BES-2013-063573) contracts for graduate students, respectively. Funding from MINECO is partially contributed by the European Regional

Author contributions

M.B. and S.R-F. carried out the experiments, analyzed the data generated, and contributed to artwork design. X.R.B. conceived the work, analyzed data, wrote the manuscript, and carried out the final editing of figures.

Competing interests

The authors report no competing financial interests.

References (45)

  • E. Rozengurt et al.

    Protein kinase D signaling

    J. Biol. Chem.

    (2005)
  • M.J. Caloca et al.

    Mechanistic analysis of the amplification and diversification events induced by Vav proteins in B-lymphocytes

    J. Biol. Chem.

    (2008)
  • X.R. Bustelo

    Vav family exchange factors: an integrated regulatory and functional view

    Small GTPases

    (2014)
  • P. Crespo et al.

    Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product

    Nature

    (1997)
  • X.R. Bustelo

    Vav proteins, adaptors and cell signaling

    Oncogene

    (2001)
  • J. Robles-Valero et al.

    A paradoxical tumor-suppressor role for the Rac1 exchange factor Vav1 in T cell acute lymphoblastic leukemia

    Cancer Cell

    (2017)
  • J. Robles-Valero et al.

    Rho guanosine nucleotide exchange factors are not such bad guys after all in cancer

    Small GTPases

    (2018)
  • J. Wu et al.

    A functional T-cell receptor signaling pathway is required for p95vav activity

    Mol. Cell. Biol.

    (1995)
  • F. Macian

    NFAT proteins: key regulators of T-cell development and function

    Nat. Rev. Immunol.

    (2005)
  • N. Movilla et al.

    Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins

    Mol. Cell. Biol.

    (1999)
  • O. Ksionda et al.

    Mechanism and function of Vav1 localisation in TCR signalling

    J. Cell Sci.

    (2012)
  • X.R. Bustelo et al.

    Vav family

  • View full text