La rata taiep como un modelo de narcolepsia-cataplejía

The known three dimensional structures of liganded and unliganded SH3 domains display few ligand-dependent differences, ruling out major conformational changes within the domain. Upon ligand binding however, the binding event juxtapositions the SH3 domain containing protein and the proline-rich ligand containing protein which may have effects on the activity of one or both proteins. The physiological role of SH3-mediated interactions is still poorly understood, but recent studies suggest that SH3 domains mediate critical protein-protein interactions. These interactions are used to organise protein complexes within the cell, bring substrates to enzymes, and regulate enzymatic activities, as discussed in the following section.

Numerous oncogenes have been isolated from acutely transforming retroviruses including the adaptor protein, Crk, and the non-receptor tyrosine kinases, Abl and Src. Mutation of the SH3 domain of the viral form of Crk (v-Crk) mitigates its transforming

potential ((Mayer and Hanafusa, 1990)). Mutational analysis of the SH3 and SH2 domains of v-Src and c-Src suggest a role for these domains in the regulation of kinase activity and in the induction of morphological changes associated with transformation (Cross et al., 1984; Erpel et at., 1995; Hirai and Varmus, 1990) (section 1.3.3.1.4). Studies of c-Abl have shown that either deletion of or point mutations within the SH3 domain result in the constitutive tyrosine kinase activity and oncogenic activation (reviewed in Wang, 1993). Unlike c-Src protein, c-Abl is not regulated by binding of its SH2 domain to a carboxy terminal phosphorylated tyrosine (section 1.3.3.1). Instead, mutational analysis has implicated the SH3 domain as a potential regulator of c- Abl kinase activity (Franz et al., 1989; Jackson and Baltimore, 1989; Van Etten etal.,

1995) The SH3 domain of c-Abl is thought to normally interact with a transregulator which blocks the kinase activity (Jiangyu and Shore, 1996) The HIV protein, Nef, was found to contain a PXXP motif that mediates its interaction with the SH3 domains of Hck and Lyn (Saksela et al., 1995). The PXXP motif is required for the higher replicative potential of Nef-bearing viruses, suggesting that the virus uses this SH3- domain mediated interaction to regulate a signalling pathway that facilitates viral growth.

Another type of regulatory mechanism is found in proteins involved in superoxide generation in neutrophils. Two proteins required for activation of a mitochondrial oxidase, p67P^°^ and p47P^o^, contain two SH3 domains each. Genes encoding components of this pathway are often mutated in patients with hereditary chronic granulomatous disease (COD), who have neutrophils lacking the capacity to synthesise superoxide. Activation of the superoxide-producing by treating neutrophils with arachidonic acid or sodium dodecyl sulphate causes the SH3 domain of p67P*^°*, to bind to the proline-rich tail of p47P^°^, whose SH3 domains binds in turn to p22Ph°^, a membrane-bound component of the oxidase complex (Finan et a i, 1994; Sumimoto et al., 1994). Full activation requires both the SH3 domain and the small GTP-binding protein, Rac. (Abo et al., 1991; Clark et al., 1990). One mutation found in a CGD patient maps to the proline-rich region of p2 2P*^ox and abolishes binding to p47Pi^°^.

Presumably, the molecule is normally held in an inactive conformation by intramolecular interactions of SH3 domains with prohne-rich sequences, and these must be disrupted before the active complex can be assembled.

Other examples of the regulation of enzymes by SH3 domain-containing proteins are the activation of PI3K by binding of SH3 domains to its 85 kDa subunit (Pleiman et al.,

1994) (discussed in Chapter 5), and the increase in the GTPase activity of the endocytic protein dynamin that is observed upon binding of various SH3 domains (Gout et al.,

(Collins, 1991). Mammalian dynamins have approximately 80% homology to the

Drosophila melanogaster shibire gene (van der Bliek and Meyerowitz, 1991). Shi^^

flies were initially isolated as temperature-sensitive mutants (Grigliatti et at., 1973) that were paralysed as a result of depletion of neurotransmitter-containing vesicles at nerve terminals (Poodry et a i, 1993). Similar mutations in the GTP-binding domain of mammalian dynamin inhibit receptor-mediated endocytosis (Herskovits et at., 1993; van der Bliek et al., 1993). The enzymatic activity of dynamin is markedly increased through association with microtubules (Herskovits etal., 1993; Shpemer and Vallee,

1992), acidic phospholipids, in particular PtdIns(4,5)P2 (Salim et al., 1996; Tuma et al., 1993), and certain regulatory proteins that contain SH3 domains (Gout et al., 1993; Herskovits et al., 1993). A study using affinity chromatography showed that dynamin binds selectively to the SH3 domains of PLCy , Grb2 and p85a, but not to several other SH3 domains. Dynamin has consensus SH3 domain binding sequences located at its carboxyl terminus. Furthermore, fusion proteins of Grb2, Src and p85a SH3 domains were able to stimulate the GTPase activity of brain dynamin (Gout et al.,

1993). It is as yet unclear whether SH3 domain binding regulates dynamin GTPase activity in vivo. There is evidence that the activity of dynamin can also be modulated by phosphorylation. Protein kinase C (PKC) can phosphorylate dynamin, leading to stimulation of its GTPase activity, whilst the calcium-dependent phosphatase, calcineurin, can reverse this stimulation (Liu et al., 1994; Liu et al., 1994; Robinson et al., 1993). Interestingly, the PKC phosphorylation site is located within the proline- rich regions of dynamin, and phosphorylation and dephosphorylation of these sites correlates with the polarisation and depolarisation of synapsosomes at the nerve terminals (Liu et al., 1994). Whether these phosphorylation events involve or regulate SH3 domain-binding has yet to be investigated.

Compartmentalisation of proteins within the cell plays an important role in the regulation of signal transduction processes. Many SH3 domain-containing proteins associate with the cytoskeleton or actin filaments, including a-spectrin (Wasenius et al.,

1989), myosin-1 (Jung et al., 1987) and cortactin (Wu and Parsons, 1993). Genetic analysis in yeast has demonstrated that SH3-domain containing proteins such as ABP- 1, SLAl, BEMl and BEM2 are required for the organisation and polarisation of the cytoskeleton (Kavanaugh et al., 1994). In D.melanogaster, mutations in the tumour suppressor gene, discs large (dig), leads to a loss of the tight septate junctions between epithelial cells, and aberrant proliferation of cells in the imaginai disk (Woods and Bryant, 1989). In mammalian fibroblasts, the SH3 domains of Grb2 and PLCyl localise to membrane ruffles and actin stress fibres respectively, suggesting a role for SH3 domains in the control of protein distribution within the cell (Bar Sagi et al.,

The interaction between the SH3 domain of a-spectrin and the proline-rich carboxy- terminal tail of the amiloride-sensitive Na'*' channel dictates the localisation of a spectrin to the apical membrane of polarised epithelial cells (Rotin et al., 1994).

Thus, SH3 domains can fulfil a variety of roles in the regulation of enzyme activity, the assembly of multiprotein complexes or specification of protein localisation within the cell.