Capítulo 4. Las Redes Sociales como sustrato del constructo de Interculturalidad
4.1 Conceptualización de la Identidad Social
(v) overexpression of murine PtdlnsPK Ip can rescue temperature-sensitive M SS4 mutants (Desrivieres et a l, 1998; Homma et a i, 1998). These data indicate that MSS4 is a functional homologue of the type I subfamily of PtdIns(4)P 5-kinases (Desrivieres et a l, 1998; Homma et a l, 1998). Type II PtdIns(5)P 4-kinase activities are not known in lower eukaryotes and the absence of a type II isozyme in yeast suggests it is doubtful that the PtdIns(5)P —> PtdIns(4,5)P2 pathway exists in yeast.
Although yeast genetics provides a powerful system for the study of PI pathways relating to vesicle traffic and cytoskeletal regulation, this organism lacks certain important signalling pathways of higher eukaryotes, most notably the receptor-dependent PI 3- kinase and PLCp/y pathways. This is likely to be due to the complex signalling machinery required in the multicellular organism. The D. melanogaster and C. elegans genomes have recently been found to contain putative PtdlnsPKs which may prove to be useful genetic models for the dissection of PtdlnsPK function in metazoan organisms.
1 . 5 . 3 P h o s p h o i n o s i t i d e 3 - k i n a s e s
Evidence for the existence of biosynthetic pathways involving Ptdlns phosphorylation at the D-3 position was initially provided by the discovery that Ptdlns 3-kinase activity co precipitated with various PTKs including activated PDGFp (Whitman e ta l, 1985), v-Src and the middle-T antigen from polyoma virus-infected cells (Courtneidge and Heber, 1987). Soon after it was found that a novel lipid, PtdIns(3,4,5)P3, was rapidly synthesised on activation of neutrophils (Traynor Kaplan et a l, 1989). This work led to the purification and subsequent cloning of the 110 kDa catalytic subunit of bovine PI 3- kinase (Hiles et a l, 1992; Morgan et a l, 1990), the first mammalian member of an enzyme family that has expanded rapidly in recent years and has been the subject of intensive study. The characterisation of the PI 3-kinases has established diverse cellular roles including regulation of cell survival (Franke et a l, 1997), transformation (Chang et a l, 1997), and membrane traffic (reviewed by Shepherd et a l, 1996). This Section will not attempt to give a comprehensive review of the PI 3-kinase literature but instead a summary of PI 3-kinase structure and function (for review see Toker and Cantley, 1997; Fruhman et al, 1998).
1 . 5 . 3 . a The PI 3-kinase g e n e family
The PI 3-kinases have been classified according to their structure, lipid substrate specificities, and their mode of regulation (Domin and Waterfield, 1997). These are summarised in Figure 1.8.
Figure 1.8 The PI 3-kinase fam ily
Summarised properties of the PI 3-kinases (see Section 1.5.3 for details). Pi's known to act as substrates in vitro are indicated. Definitions are as follows: p i 10a, p, and ô, all mammalian 110 kDa class la PI 3Ks (see 1.5.3.a for relevant references); AGE-1 C. elegans class la PI 3K (Morris et a i, 1996); PIK l, PIK2, PIK3, D. discoideum class la and class Ib PI 3Ks (Zhou et al., 1995a); pllOy, mammalian GPCR-regulated PI 3K; Cpk-m/pl70, murine (Molz et a l, 1996; Virbasius et a l, 1996); PI3K-C2a, P, and y human class II PI 3K isoforms (Domin et a l, 1997; Brown et a i, 1997; Ono et al.,
1998); 68D, D. melanogaster class II PI 3K (MacDougal et a i, 1995); Vps34, S. cerevisiae VPS34 gene product (Shu et a i, 1993); hVps34, human Vps34p homologue (Volinia et a i, 1995).
Class
Structure Isoform Adaptor Regulation Ptdlns, Ptdlns(4)P, Ptdlns(4,5)P2 p110a,p,5 Dp110 AGE-1 PIK1,2 p85a,p p55a,Y p 5 0 a p60 Tyrosine kinases Ras B p110y PIK3 plOI p 1 2 0G-protein coupled receptors Ras
II
Ptdlns, Ptdlns(4)P p r o l i n e - r i c h Cpk-m/p170 PI3K-C2 a ,(3,Y 68D unknown unknownIII
Ptdlns Vps34 hVps34 VpsIS pi 50 unknown k in a s e domain Ras i n t e r a c t i o nPIK dom ain (LKH2)
a d a p to r - b in d in g
C2 domain
UJ VC
The class I enzymes can phosphorylate Ptdlns, PtdIns(4)P, PtdIns(5)P and Ptdlns(4,5)?2 vitro (Serunian et al., 1989). Agonist stimulation of these enzymes in vivo increases cellular levels of PtdIns(3,4)P2 and P tdlns(3,4,5)?3, although PtdIns(3,4)P2 can be generated by PtdIns(3)P 4-kinase activity (Banfic et al., 1998b), or by PtdIns(3,4,5)P3 (5)-phosphatase activity (Stephens et al., 1991), both of which may contribute to this response. Four class I enzymes have been characterised in mammalian systems, p i 10a, pilO p, pllO y and pi 105 (Hiles et al., 1992; Hu et al., 1993; Stoyanov et al., 1995; Vanhaesebroeck et al., 1997). p i 10-related genes have also been cloned from a range of eukaryotes including C. elegans, D. discoideum and D. melanogaster (Leevers et al., 1996; Morris et al., 1996; Zhou et al., 1995a). All members of this class form heterodimeric complexes with regulatory subunits that couple these enzymes to upstream signalling systems (reviewed in Fruhman, 1998). PI 3-kinases 110a, -p, and -5 (class la) are able to interact with the p85 family of adaptor subunits, p85a, p50a, p55a, p85p, and p85y which arise from three distinct genes (Antonetti et al., 1996; Otsu et al., 1991; Pons et a l, 1995). p i lOy (class Ib) interacts with an unrelated regulatory sunbunit, plOl (Stephens et al., 1997). The p85 family lack intrinsic enzyme activity but contain SH2, SH3 and proline-rich domains which have the capacity to interact with multiple proteins. It has not been shown that different Class I PI 3-kinases have any preference for individual adaptor subunits (Vanhaesebroeck et al., 1997), however, different p85 isoforms may interact with distinct sets of intracellular proteins (Baltensperger et al., 1994; Reif et al.,
1993; Shepherd et al., 1996).
In the case of the class Ia enzymes, adaptor subunits regulate subcellular localisation and activity: many agonists stimulate the phosphorylation of tyrosine residues generating potential binding motifs for proteins containing SH2 domains. The SH2 domains of p85 have been studied in detail and the use of synthetic peptides has been used to determine the binding specificities of p85 SH2 domains. Both SH2 domains bind preferentially to peptides containing pYXXM motif and an additional methionine or valine C-terminal to the phosphotyrosine residue increases binding affinity (Sonyang et al., 1993). This YXXM sequence can be found in many proteins that are known to activate heterodimeric PI 3- kinase for example, polyoma middle T antigen (Druker et al., 1990; Ling et al., 1992) and peptides corresponding to PDGF receptor autophosphorylation sites bind to p85 proteins with high affinity in vitro, and stimulate the activity of the associated catalytic subunits (Backer et al., 1992; Carpenter et al., 1993). Recruitment to activated RTKs brings PI 3- kinase to the plasma membrane and also activates the enzyme towards PtdIns(4,5)P2- The importance of translocation is demonstrated by the observation that constitutively targeting the p i 10 subunit to the plasma membrane is sufficient to elevate levels of cellular PtdIns(3,4,5)P3 (Klippel et al., 1996). Ras interaction may also recruit heterodimeric PI 3-kinase to the membrane in a similar way to Raf (Leevers et al., 1994) or alternatively membrane recruitment may bring PI 3-kinase to membrane-bound Ras.
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