CÁMARAS DE SEGURIDAD

ppôO^^'^^^, the product of the first molecularly defined proto-oncogene and first protein tyrosine kinase discovered, has provided a paradigm for the understanding of PTKs. There are currently eight other members of the Src gene family; Fyn, Lyn, Yes, Lck, Blk, Fgr, Hck and Yrk (Cooper and Howell, 1993; Sudol et al., 1993). This family of kinases are structurally similai* over all but the first 60-80 amino acids of their total length of

500-530 residues. The conserved regions can be divided into five sequence blocks; the extreme N-terminal myristoylation signal, the SH3 and SH2 regions, the kinase domain and the C-terminal non-catalytic tail. These proteins also contain two conserved tyrosine residues, one within the kinase domain and the other in the C-terminus, which are both targets for phosphorylation.

Regulation o f pp60^'^^^

The Src family all have a conserved tyrosine residue within their kinase domain (Tyr 416 in pp60^'^^^). ppbO^ ®’^^ autophosphorylates at Tyr 416 and is phosphorylated in vivo at a tyrosine residue in the C-terminal tail (Tyr 527) by the tyrosine kinase Csk (C-terminal Src kinase) (Cooper et al., 1986; Courtneidge, 1985). The oncogenic form, pp60''-^^^, lacks Tyr 527. Mutation of this residue to phenylalanine increases the protein-tyrosine kinase activity of pp60^*®’‘‘^ and induces its transforming potential (Kmiecik and Shalloway, 1987; Piwnica Worms et al., 1987). Concomitant with either the lack of or underphosphorylation of Tyr 527 is hyperphosphorylation of Tyr 416 in the catalytic domain. In vitro experiments showed that the C-terminal tail phosphorylation is inhibitory and the kinase domain phosphorylation is stimulatory for the tyrosine kinase (Kmiecik and Shalloway, 1987). As the region containing the C-terminal tyrosine is protected from proteases it was suggested that it probably bound to an internal site in pp60^-^^^ (Cantley et al., 1991). Genetic and biochemical data implied that the inhibition of pp60^'^^^ kinase activity, induced by phosphorylation of Tyr 527 (Cooper et al., 1986; Courtneidge, 1985), required a physical interaction between the SH2 domain and the tyrosine phosphorylated tail (Amrein et al., 1993). When overexpressed, Csk inhibits transformation of fibroblasts induced by high levels of ppbO^^'^^^^ activity (Sabe et al.,

1992). Mutant mice lacking the csk gene die at the neurulation stage, and cell lines established from these mice have increased phosphotyrosine levels and increased activity of Src family kinases (Imamoto and Soriano, 1993; Nada et al., 1993).

The recently published crystal structures of pp60^'^^(^ (Xu et al., 1997) and Hck (Sicheri et al., 1997) kinases, have provided conformation for the above model of kinase repression, but also revealed additional novelties in their regulation (Pawson, 1997). The crystal stmctures of PTKs, ppbO^^'^^^ and Hck demonstrate the intramolecular reactions involved in kinase repression. In both structures the SH2 domain is bound to the C-terminal tail as in the above model. Moreover, the structures reveal how the SH3 domain is a key module in contributing to the stability of the repressed state. Unexpectedly, the linker connecting the SH2 domain to the catalytic domain, forms a polyproline type II helix and can bind to the SH3 domain. In Hck, the SH3 domain contacts the small lobe of the

kinase domain thereby inhibiting its activity through conformational constraints on the catalytic domain (Pawson, 1997). In the pp60^'®^^ structure, these intramolecular interactions involving the SH2 and SH3 domains, were shown to result in an inactive conformation, and prevent the PTKs SH2 and SH3 binding surfaces from interacting with other proteins. It seems that a key function of the SH2 domain-pTyr interaction may be to position the SH3 domain so that it can bind to the SH2-kinase linker, and consequently contact the small lobe of the kinase domain.

Activation

Activation of pp60^'^^^ can therefore be achieved either by dephosphorylation of the C- terminal tyrosine by a phosphatase, which would result in an open conformation, or by disruption of the intramolecular SH2/SH3 binding by competitive higher affinity interactions. The transmembrane glycoprotein CD45 possesses protein tyrosine phosphatase activity and has been shown to activate Lck and Fyn in T lymphocytes. Moreover, in CD45 deficient cells, Tyr-505 of Lck was hyperphosphorylated, and Fyn phosphorylation and kinase activity were also altered (Mustelin and Bum, 1993). Other PTPs that activate Src kinases are also presumed to exist in other cell types.

High affinity SH2 domain-interactions can also displace the C-terminal tail of pp60^*^^^ and stabilise its active conformation. Intermolecular binding of ppbO^^*^^^ SH2 domain to the phosphorylated tyrosines of the PDGF receptor, disrupts the intramolecular binding of the SH2 domain to its pTyr-527, and the activities of ppôO^^'®^^, c-Yes and Fyn all increase following association with ligand-stimulated PDGF receptor (Twamley et al., 1992; Twamley Stein et al., 1993). Phosphorylated peptides that bind more tightly to the ppbO^’^*"^ SH2 domain than its C-terminal phosphotyrosine (Songyang et al., 1993), also increase its kinase activity (Liu et al., 1993). Deletion or mutation of the SH2 domain can also convert ppbO^^'®^^^ to a transforming protein (Seidel Dugan et al., 1992).

Recent data have shown that competition for the kinase's SH3 domain interaction is also an important mechanism for activation, since binding of the HIV-1 Nef protein to the SH3 domain of Hck causes a dramatic increase in kinase activity (Moarefi et al., 1997; Sicheri et al., 1997). The binding of Nef to the SH3 domain dismpts the SH3 domain- linker interaction, which perturbs the inactive conformation of the kinase. Nef stimulates the activity of Tyr-527 phosphorylated Hck and also further increases the activity of dephosphorylated Hck. Binding of Nef to the SH3 domain of Hck also promotes autophosphorylation of the catalytic domain tyrosine residue in trans, which is thought to lift some constraint on the kinase domain and increase its activity (Moarefi et al., 1997).

Biological roles o f Src Kinases

The Src family kinases have been shown to interact directly with a number of intracellular signalling molecules. These include PI 3-kinase (Pleiman et al., 1993; Sugimoto et al., 1984; Taichman et al., 1993; Vogel and Fujita, 1993; Whitman et al., 1985), RasGAP (Brott et al., 1991; Ellis et al., 1990), Raf-1 (Morrison et al., 1988), PLC Y (Nakanishi et al., 1993a), Nck (Chou et al., 1992; Meisenhelder and Hunter, 1992) and She (McGlade et al., 1992). A requirement for the Src family kinases in mitogenesis has been demonstrated in vivo. Microinjection of DNA encoding catalytically inactive forms of and Fyn proteins inhibits PDGF-stimulated entry of cells into S-phase (Twamley et al., 1993). In addition, deletion of the src gene in osteoclasts results in cells unable to reabsorb bone efficiently and thus osteopetrosis (Soriano et al., 1991). The op

mutation also causes osteopetrosis and is contained in the gene encoding CSF-1. This suggests that CSF-1 may be required for differentiation of mature osteoclasts acting through the CSF-1 receptor which binds and activates pp60^'^^(^ (Courtneidge et al.,

1993).

ppôO^-^'^^ has also been linked to the integrin-mediated FAK pathway, through its ability to phosphorylate and activate the focal adhesion kinase (FAK), necessary for the attachment of cells to extracellular matrix (Cobb et al., 1994). Finally, ppôO^'®^'^ has been found to localise at endosomes and microtubule structures in fibroblasts, possibly suggesting a role in protein trafficking and mitotic centriolar organisation (David-Pfeuty and Nouvian-Dooghe, 1990; Kaplan et al., 1992).