| Regulation of cell-cell adhesion and actin cytoskeleton in non-transformed and transformed epithelial cells | ||
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Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, differentiation and survival. Several families of receptor and non-receptor tyrosine kinases control these events by catalysing the transfer of phosphate from ATP to a tyrosine residue of specific cell protein targets.
Transformation is the process that in animals leads to formation of tumours and in cell culture induces structural, biochemical and behavioural changes in the cells. The prototype member of the Src family protein tyrosine kinases was originally identified as the transforming protein (v-Src) of the oncogenic retrovirus, Rous sarcoma virus, RSV (Brugge & Erikson 1977, Purchio et al. 1978, Hamaguchi et al. 1995). Viral v-Src is a mutated and activated version of a normal cellular protein (c-Src) with intrinsic tyrosine kinase activity (Collett & Erikson 1978, Levinson et al. 1978). This kinase phosphorylates its protein substrates exclusively on tyrosyl residues (Hunter & Sefton 1980). In epithelial cells it is associated with adherens junctions and focal contacts suggesting a role in cell adhesion (Tsukita et al. 1991, reviewed by Frame et al. 2002).
Src protein tyrosine kinases are 52-62 kD proteins composed of six distinct functional domains: SH4 (src homology 4), a unique domain, SH3, SH2, SH1 and a C-terminal regulatory region (reviewed by Brown & Cooper 1996). SH4 domain contains the myristylation signals that guide the Src molecule to the cell membrane. This unique domain of Src proteins is responsible for their specific interaction with particular receptors and protein targets (reviewed by Thomas & Brugge 1997). Moreover, it binds to atypical protein kinase C (Seibenhener et al. 1999). The modulating regions, SH3 and SH2, control intra- as well as intermolecular interactions with protein substrates which affect Src catalytic activity, localisation and association with protein targets (reviewed by Pawson 1995). The kinase domain, SH1, found in all proteins of the Src family, is responsible for the tyrosine kinase activity and has a central role in binding of substrates. The N-terminal half of Src kinase contains the site(s) for its tyrosine phosphorylation, and phosphorylation of tyrosine (Y) 416 regulates the catalytic activity of Src (reviewed by Thomas & Brugge 1997). v-Src differs from cellular Src (c-Src) on the basis of the structural differences in C-terminal region responsible for regulation of kinase activity. V-Src always exists in opened, active conformation, whereas c-Src is flexible and normally inactive (reviewed by Thomas & Brugge 1997). Activation of c-Src is critically involved in carcinoma cell migration and metastasis (Sakamoto et al. 2001).
Src substrates are proteins that become tyrosine phosphorylated as a result of src gene function and are direct or indirect targets of Src. The best-known Src substrates are found in transformed cells. Most of them, found at focal adhesions, are key components in the integrin-mediated signal transduction and bound to actin or integrin, e.g. vinculin, cortactin, talin, paxillin, FAK, tensin, ezrin and p130cas. Furthermore, junctional proteins, such as β - and γ -catenin, ZO-1, occludin, p120ctn, connexin 43, nectin-2 delta are identified as major sites of tyrosine phosphorylation by Src kinases (reviewed by Parsons & Parsons 1997, reviewed by Thomas & Brugge 1997, Tsukamoto & Nigam 1999, reviewed by Abram & Courtneidge 2000, Kikyo et al. 2000). The intercellular communication via gap junctions is prevented when two tyrosines of connexin 43 are phosphorylated by v-Src (Lin et al. 2001). The other known targets of Src are enzymes involved in phospholipid metabolism, such as PLC-γ , p85 subunit of PI3-kinase and the signalling molecules p190RhoGAP, p120rasGAP and EGF receptor substrate, Eps8 (reviewed by Thomas & Brugge 1997, reviewed by Abram & Courtneidge 2000, Gallo et al. 1997).
The regulation of cellular adhesion by v-Src kinase is still poorly understood. It is known that Src has an essential role in bone formation, since its mutation induces defects in bone remodelling of mice including impaired osteoclast function and developed osteopetrosis (Soriano et al. 1991). Experiments with Src-/- fibroblasts have shown that Src is not essential for the formation of focal adhesions but it is required for the optimal adhesion and spreading on fibronectin matrix mediated by integrin receptors (Felsenfeld et al. 1999, Cary et al. 2002). Attachment to fibronectin stimulates phosphorylation of focal adhesion kinase (FAK) induced by integrin engagement and activated Src (McLean et al. 2000). In MDCK cells low expression of v-Src predominantly disturbs the formation of adherens junctions but has no effect on the assembly of tight junctions and desmosomes (Warren & Nelson 1987). Moreover, in v-src transformed MDCK cells activation of v-Src weakens the cadherin-based adhesion (Volberg et al. 1992, Behrens et al. 1993, Takeda et al. 1995). The tyrosine phosphorylation of the cellular proteins (especially proteins of zonula adherens) such as β -catenin is increased in these cells, correlating with their dedifferentiation and promotion of the invasiveness (Takeda et al. 1995, reviewed by Noe et al. 1999). In src-MDCK cells activation of v-Src also releases spectrin from the cell membranes (Sormunen et al. 1999). To date it is unclear whether v-Src or c-Src interacts directly with the components of adherens junctions in vivo. Moreover, tyrosine phosphorylation of β -catenin in response to growth factors and cell transformation is not connected with the shift of adhesion from strong to weak (Takeda et al. 1995). Although p120ctn was first identified as a Src substrate and its constitutive tyrosine phosphorylation correlates with cell transformation, it is not clear what is the significance of tyrosine phosphorylation of p120ctn and how it affects on Rho or cadherin function (Anastasiadis & Reynolds 2000). However, in Ras-transformed breast epithelial MCF10-A cells p120ctn is heavily phosphorylated, adherens junctions are loosened and p120ctn is efficiently bound to cadherin (Kinch et al. 1995). Expression of cadherin also controls recruitment of p120ctn to cell-cell contacts and association of p120ctn to E-cadherin is a prerequisite for tight adhesion (Thoreson et al. 2000).