3. Introduction
3.3. Components of the ITAM(-like) signalling pathway
3.3.2. Adaptors and scaffolds
After phosphorylation of the tyrosines in the ITAM-like sequence of CEACAM3, multiple interactions are possible. While several kinases themselves can also bind to CEACAM3 via their SH2 domains, additional factors are recruited in response to tyrosine phosphorylation. For one, the guanine nucleotide exchange factor (GEF) Vav1 can directly bind to pY230 of CEACAM3. In common immunoreceptor signalling, Vav is recruited more indirectly to promote activation of the small GTPase Rac by catalysing the exchange of Rac bound GDP for GTP. Nck is also recruited to this signalling complex via its SH2 domain and localises Rac's effector in terms of actin polymerisation: the WAVE-complex.
3.3.2.1. The Vav-family of guanine nucleotide exchange factors Vav is a protein with numerous
protein-protein-interaction sites. In humans, there are three isoforms of Vav, which are identical in regard of domain structure. Differences arise in the expression patterns, with Vav1 being restricted to the hematopoietic system and Vav2 and Vav3 being expressed in a wider range of tissues. Vav could be grouped into the effector category, since its known function makes it an effector of CEACAM3, that relays
the activation to the actin polymerisation machinery by activating a key regulator, the small GTPase Rac by acting as a guanine nucleotide exchange factor (GEF). But keeping in mind Vav's complex assembly that shows several protein-protein binding motifs and interaction domains, including two SH3 and one SH2 domain along with one PH and one CH domain, justifies grouping it into the scaffolding category. Vav can be phosphorylated on several tyrosine residues, allowing further interactions with SH2-domain proteins. A proline rich region allows binding of SH3 domains, and two more SH3 domains within the c-terminus of the Vav protein enable recognition of proline rich stretches in other proteins. A SH2 domain is embedded in between those SH3 domains and confers binding to phospho-tyrosines, as in our field of interest, to phosphorylated tyrosine 230 (pY230) in CEACAM3 (Schmitter et al., 2007a).
Figure 3.3.4: The guanine nucleotide exchange factor (GEF) Vav is implicated in many processes. A multitude of domains facilitates interactions with phospholipids, phosphoproteins and proline-rich sequences, making Vav an expedient scaffold, too (Tybulewicz 2005)
Introduction
Components of the ITAM(-like) signalling pathway
Recently the structural and mechanistical properties of Vav inhibition and activation were investigated, revealing a highly cooperative intramolecular network of interactions regulating GEF activity, that depends on the DH-domain adjacent acidic helix and the CH domain. Additionally, activation needs tyrosine phosphorylation on Y142, Y160 and Y174, and occurs in that order, since Y174 is not readily accessible without prior phosphorylation of the other tyrosine residues (Yu et al., 2010).
Tyrosine 174 is phosphorylated by the Src-PTK Lck in vivo, and is crucial for T-cell development and activation (Miletic et al., 2006). The assembly after T-cell receptor (TCR) stimulation is quite complex, involving several adapter proteins like LAT, SLP76 and Vav. Vav is a versatile signalling protein in T-cells (reviewed in (Tybulewicz, 2005)), acting on multiple events such as calcium flux, development and actin reorganisation. Vav proteins are needed for generation of the oxidative burst in macrophages and neutrophils, whereas FcR-mediated phagocytosis only needs Vav in neutrophils, but not in macrophages (Utomo et al., 2006). Vav is involved in actin cytoskeleton reorganisation in multiple scenarios (reviewed in (Hornstein et al., 2004)). It functions in formation of the T-cell signalling complex with antigen presenting cells, termed immunological synapse, as well as in integrin mediated actions as T-cell or platelet spreading. Vav was found to be involved in FcR and complement receptor mediated phagocytosis. While its role as a Rac GEF downstream of FcR ligation (Patel et al., 2002) has been challenged (Hall et al., 2006), it was found to be essential for the phagocytosis via the complement receptor (termed CR3 or Mac1 or CD11b/CD18 or αMβ3 integrin). In the context of CEACAM3 mediated phagocytosis Vav's activity as a Rac GEF is essential (Schmitter et al., 2007a). This pathway is continued by activation of PAK, and subsequently (as also observed in context of FcεR signalling (Arudchandran et al., 2000), the activation of JNK (Hauck et al., 1998) that is found equivalently in CEACAM signalling. For all those activities, the SH2 domain of Vav is critical, and multiple proteins allowing binding of Vav via its SH2 domain have been identified so far. For T-cell signalling, the kinase ZAP70 has been shown to be an important docking partner (Katzav et al., 1994) and the tyrosine residue (Y)315 in this protein mediates this critical interaction (Wu et al., 1997). Generally, Vav may bridge phosphorylated ITAMs to further scaffolds by interaction with Syk family kinases ((Deckert et al., 1996) review in (Bustelo, 2001)) and thereby bring additional downstream effectors into place. This again is a difference to the pathway we observe in CEACAM3 signalling.
Components of the ITAM(-like) signalling pathway
cytoskeleton and other cellular responses to environmental signals, that have been studied in quite detail but nonetheless will prove interesting in the future regarding the complexities of their modes of signalling determined by selective scaffolding and/or catalytical activity.
3.3.2.2. The Nck family, ubiquitous adapter proteins Another small family of scaffolding
proteins is the family of Nck proteins.
Ncks are adaptor proteins containing three SH3 domains and one SH2 domain and have been implicated in many processes such as neuronal development (Fawcett et al., 2007;
Guan et al., 2007), invadopodia
formation (Stylli et al., 2009) and other actin cytoskeletal rearrangements. There are two isoforms of adaptor protein Nck, Nck1 and Nck2, that seem to be redundant in most scenarios. This is also true for embryonic development, where knockout of one Nck isoform allows normal development but knockout of both Nck genes results in lethality around day nine in embryonic development (E9.5) (Bladt et al., 2003). The likely redundancy in function does not come as a surprise when considering the high sequence identities of the two isoforms. Both are 68% identical on the whole-protein level, and the distinct SH domains rank even higher, with 76% in the 3rd SH3 domain being the lowest and 86% in the SH2 domain the highest degree of sequence identity (reviewed in (Lettau et al., 2009)). Nck1 and Nck2 display differences in their expression patterns, with ubiquitous expression of Nck1 and more restricted expression of Nck2 (Bladt et al., 2003). Although differences in binding affinities of the SH2 domains were described, specificities vary only insignificantly (Frese et al., 2006) reflecting the conservation of the tyrosine-peptide interacting residues in both isoforms.
But also the SH3 domains have been implicated in many protein-protein interactions.
Since the canonical SH3-binding motifs (proline rich sequences) usually do not require enzymatic modification to assume a recognisable conformation, these interactions are considered to be constitutive and regulated only by accessibility of the binding sequence. And indeed, a recent study detected an inhibitory
Figure 3.3.5: The adaptor protein Nck exists in two isoforms, each consisting of three SH3 domains and one SH2 domain, that share large sequence identity in these domains (Lettau 2009)
Introduction
Components of the ITAM(-like) signalling pathway
intramolecular interaction in Nck2, that modulated its ability to bind to proline rich sequences in trans by masking the binding site of the 2nd SH3 domain through an adjacent arginine rich stretch in Nck2 itself (Takeuchi et al., 2010).
Already ten years ago numerous interaction partners for Nck had been described (reviewed in (Li et al., 2001)), including a multitude of actin cytoskeleton related proteins. High local
concentrations of Nck SH3 domains trigger actin polymerisation via N-WASP in an artificial system (Rivera et al., 2004) without the need for Cdc42 activity. In a more physiological setting, Nck initiates actin polymerisation via N-WASP synergistically with PI(4,5)P2 (Rohatgi et al., 2001; Tomasevic et al., 2007). Besides binding to WASP via its 3rd SH3 domain, Nck can also bind via its 1st SH3 domain to Nap1, which is a constituent of the Wave-complex, another activator of the Arp2/3-complex (Kitamura et al., 1996).