New insights into the Vav1 activation cycle in lymphocytes
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.
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