**2. p130Cas adaptor protein**

#### **2.1 p130Cas adaptor features**

p130Cas is coded in human by the BCAR-1 (Breast Cancer Anti-oestrogen Resistance 1) gene. This gene is conserved through many species and in humans is localized on Chromosome 16q23.1. Knock-out of the mouse gene results in embryonic lethality at 9.5 days, indicating that any other protein cannot fill in for its role during development. p130Cas is an ubiquitously expressed multi-site docking protein that consists of i) an Nterminal Src homology 3 (SH3) domain, ii) a substrate domain, which contains 15 repeats of a YXXP sequence (tyrosine-any two aminoacids-proline), iii) a serine rich region, and iv) a C-terminal domain (Figure 1A). The presence of these multiple conserved sequence motifs and extensive post-translational modification, mainly consisting of tyrosine and serine phosphorylation, allow the assembly of specific multi-protein complexes. In particular, the SH3 domain interacts with polyproline-rich sequences present in several proteins including

human tumours leading to uncontrolled dissemination of cancer cells into the body (Berx *et* 

At least three major classes of membrane proteins are involved in these events, namely, the E-cadherin, the Receptor tyrosine kinases (RPTKs), and the integrin receptors. The cell-cell adhesion receptor E-cadherin is the major membrane protein involved in binding between neighbouring cells in adherens junctions. As a practical consequence of its adhesive functions, E-cadherin has also been shown to prevent EGFR activation and downstream signalling, leading to negative regulation of proliferation (Berx and Van Roy, 2001; Gutkind, 2000; Perrais *et al.*, 2007; Qian *et al.*, 2004). E-cadherin is frequently down-regulated or lost in epithelial tumours, and its loss correlates with increased cancer cell invasiveness ((Peinado

Integrins are cell surface heterodimeric receptors for the ECM formed by the non covalent association of alpha and beta subunits (Hynes, 2004). Integrins specifically localize to focal adhesions, which are sites of close apposition with the ECM where actin filaments are anchored to the plasma membrane. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors or growth factor and cytokine receptors, thus regulating integrin ability to transduce signals inside the cells, the so called "outside-in signalling" ( Cabodi *et al*., 2010). A growing body of evidence shows that integrins, RPTKs and cytokine receptors have no longer to be considered as individual receptors, but rather as joint modules in which attachment to the matrix confers positional control to respond to soluble growth factors (Cabodi *et al*., 2010b; Cabodi *et al.*, 2008; Desgrosellier *et al.*, 2009; Streuli, 2009; Uberti *et al.*). In the case of the EGF receptor (EGFR), beta1 integrin is both sufficient to partially activate the receptor itself and required for the full activation of the EGFR in response to EGF (Morello *et al.*; Moro *et al.*, 1998)). Integrin-dependent EGFR trans-activation accounts for a specific repertoire of mechanisms, namely cell survival and actin cytoskeleton

In this chapter we will focus on p140Cap and p130Cas adaptors as major regulators of cell migration and invasion (Cabodi *et al.*). Owing to their modular structure, both proteins can undergo tyrosine phosphorylation and association with effector proteins, leading to the assembly of molecular platforms that regulate the variety of signalling events originating

p130Cas is coded in human by the BCAR-1 (Breast Cancer Anti-oestrogen Resistance 1) gene. This gene is conserved through many species and in humans is localized on Chromosome 16q23.1. Knock-out of the mouse gene results in embryonic lethality at 9.5 days, indicating that any other protein cannot fill in for its role during development. p130Cas is an ubiquitously expressed multi-site docking protein that consists of i) an Nterminal Src homology 3 (SH3) domain, ii) a substrate domain, which contains 15 repeats of a YXXP sequence (tyrosine-any two aminoacids-proline), iii) a serine rich region, and iv) a C-terminal domain (Figure 1A). The presence of these multiple conserved sequence motifs and extensive post-translational modification, mainly consisting of tyrosine and serine phosphorylation, allow the assembly of specific multi-protein complexes. In particular, the SH3 domain interacts with polyproline-rich sequences present in several proteins including

from the complex cross-talk among integrins, E-cadherin and RPTKs.

*al.*, 2007; Cavallaro and Christofori, 2004; Giancotti, 2003; Guo and Giancotti, 2004),

*et al.*, 2007; Reynolds and Carnahan, 2004);.

organization involved in cell migration.

**2. p130Cas adaptor protein 2.1 p130Cas adaptor features** 

Fak, PYK2/RAFTK, phosphatases like PTP-PEST, PTP1B, and effectors as C3G and CIZ (Sakai *et al.*, 1994; Tikhmyanova *et al.*). The substrate domain, upon Src family kinases activation, is tyrosine phosphorylated and exposes additional binding sites for SH2 containing proteins such as the Crk adaptors (Salgia *et al.*, 1996)**,** while the serine rich region represents a docking site for other partners such as 14-3-3 and Grb2. Lastly, the C-terminus contains a polyproline-rich region responsible for the binding of the Src family kinase, PI3K, Bcar3/AND-34, Chat-H and ubiquitin ligases such as AIP4, APC/C and CDH1, as well as a binding site for the adaptor protein p140Cap (Bouton *et al.*, 2001; Cabodi *et al.*, 2004; O'Neill *et al.*, 2000).
