Material y Métodos

domain is deleted

Another way to disrupt the endogenous p85aSH3-Pl interaction is to delete the SH3 domain, therefore a deletion mutant of p85a lacking the SH3 domain was constructed as described in section 3.2.3.1. In order to determine whether deleting the endogenous SH3 domain would allow increased association with exogenous SH3 domains as would be predicted from the model described in Figure 5.7, and whether this phenomenon still occurred in the presence of the catalytic subunit, p85a and p85aASH3 were co-expressed with p i 10a using the baculovirus expression system.

GST fusion proteins of the SH3 domains from c-Src and p85a were immobilised on glutathione Sepharose CL4B and incubated with baculovirus lysates containing co­ expressed p 8 5 a /p llO a or p85aASH3/ pi 10a. In addition p 8 5 a /p l l 0 a and p85aA SH 3/pll0a were immunoprecipitated alone with an antibody directed against the SH2 domain of p85a (Lanes 1 and 4 in Figure 5.8A and B.) . In order to determine whether the exogenous GST-SH3 domains could co-precipitate the enzyme complex, the glutathione sepharose precipitates were resolved on SDS-PAGE (Figure 5.8(A). To examine whether the exogenous GST-SH3 domains could co-precipitate

lipid kinase activity, the glutathione Sepharose precipitates were subjected to PI3K assays using PI as a substrate (Figure 5.8(B)). p85aASH3/p 110a bound more efficiently than wild type p85a/pl 10a to the SH3 domains of Src and p85a (Figure 5.8A). Similarly, the GST-SH3 domains of Src and p85a could co-precipitate lipid kinase activity more efficiently from lysates containing p85aASH3/pl 10a compared to p85a/pllO a (Figure 5.8 B). It had already been shown that GST fusion proteins of the SH3 domains of p85a and Src bind efficiently to p85a PI (Figure 5.4(A)). These results show that deletion of the p85a SH3 domain allows exogenous SH3 domains access to the p85aP l region. These data support the proposed model that the p85aSH3 domain is bound to its own PI motif. Furthermore, this interaction has now been shown to occur when p85a is in complex with pi 10a.

5.3 Discussion

The modular structure of the non-catalytic p85 subunit of the PI3K has facilitated both structural and functional studies of this protein and has led to rapid advances in the understanding of its role in the heterodimeric PI3K complex. Despite these advances, the precise function of the SH3 domain in this adaptor molecule has remained elusive. In an attempt to address this question, GST fusion proteins of SH3 domains from p85a, and also from a subset of other signalling molecules, were used as affinity matrices to screen a bovine brain extract for proteins to which they could specifically bind (Gout et a/., 1993) (Figure 5.1). Several proteins were identified, including a known 100 kDa GTP-binding protein, dynamin and a 68 kDa protein (p70). (Derry et al., 1994) Phosphorylation of membrane lipids by PI3K may play a role in some of the processes involved in endocytosis, such as membrane invagination, which is known to involve dynamin (reviewed in Urrutia et at., 1997). It is possible to speculate that PI3K provides a link between transmembrane receptors which it binds through its SH2 domain, and dynamin, which it binds through its SH3 domain, leading to receptor internalisation and down regulation.

Sequence alignment of the peptides from p70 confirmed it was 50% identical to the human Wiskott Aldrich Syndrome protein (WASP) (Derry et al., 1994). Recently, a neural form of WASP, N-WASP has been identified ((Miki et al., 1996)) which is 100% identical to the tryptic peptides of p70 that were sequenced. N-WASP is an actin-depolymerising protein that regulates the cortical cytoskeletal rearrangement in a PtdIns(3,4)P2-dependent manner downstream of tyrosine kinases. Again PI3K may provide a link between PtdIns(3,4)P2, a product of PI3K catalytic activity, and the activity of N-WASP, which it binds through its SH3 domain, on the actin cytoskeleton. The involvement of PI3K in both fluid-phase endocytosis (Li et al., 1995) and

regulation of the actin cytoskeleton (Hawkins et al., 1995; Nobes and Hall, 1995) support the idea that p85a SH3 domain associates with such molecules as dynamin and N-WASP. Microinjection of the SH3 domains of Grb2 and PLCy have shown that they localise to membrane ruffles and actin stress fibres respectively. Such a study has yet to be reported for the p85a SH3 domain, but the identification of the binding partners described above supports the hypothesis that it is involved in cytoskeletal re­ arrangement and cell movement

The data presented in this chapter strongly suggest that the SH3 domain of p85a is bound to its own proline-rich motif. The first evidence came from binding studies using a biosensor. As an isolated domain, p85aSH3 was able to bind immobilised

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p85aPl peptide but not p85aP2 (compare Figure 5.4(A) and Figure 5.4(B)). In order to determine whether this interaction between p85aSH3 and p85(xPl occurred in full length p85a, mutants of p85a in which proline residues in the PI and P2 motifs were mutated to alanine were constructed and the effects of these mutations on the binding of the SH3 domain of p85a to exogenous ligands was examined. Wild type p85a

exhibited increased binding to exogenous PI peptide when endogenous p85a PI but not p85a P2 was mutated. Deletion of the SH3 domain of p85a allowed the

exogenous SH3 domains of Src and p85a to bind the endogenous p85aPl motif more efficiently (Figure 5.8). Therefore mutation of one region in p85a increased the

binding affinity of another region in the same molecule.

The best explanation for this phenomenon is an interaction of p85otSH3 and p85aPl. This hypothesis also provides an explanation for the differential binding of p85aSH3, p85aSH3BH and full length p85a described in Chapter 4. For example, p85aSH3 was shown to bind dynamin (pi00) from Jurkat and HL60 cell lysates, whereas p85aSH3BH and full length p85a did not (Figure 4.12). If p85a SH3 was bound to p85aPl in p85aSH3BH and p85a, it would be less accessible to exogenous cellular binding partners. The lack of accessibihty of the p85aSH3 domain also provides an explanation for the lack of reports of p85a binding partners in vivo.

It seems likely that p85aSH3 does not bind p85aP2. The naturally occurring isoform of p85a, p55y, which lacks the SH3 domain and PI motif but has an identical P2 sequence to p85a, could not be co-precipitated with GSTp85aSH3 (see Figure 5.11). Screening of phage display hbraries also identified p85aPl as the preferred binding partner for p85a compared to p85aP2 (Rickies et a i, 1994). It has been suggested that p85aP2 is sufficiently exposed in full length p85a to bind exogenous SH3 domains (Liu et al., 1993), although it remains possible that this sequence is occluded in full-length p85a, perhaps by steric hindrance.

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Since the studies reported in this thesis were carried out, there have been several reports which support the hypothesis that there is an intramolecular interaction between p85aSH3 and p85aPl. p85aPl is reported to be the highest affinity ligand for the p85aSH3 domain in binding experiments with synthetic peptides (Y.Zvashchenko personal communication). The structural basis for the binding of proline-rich peptides to SH3 domains has been reported (Yu et ai, 1994), (Feng et al., 1994). The use of biased combinatorial libraries, multidimensional NMR and structure based mutagenesis showed that only two proline residues in a proline-rich motif made direct contact with the SH3 domain, whilst the others appeared to function as a molecular scaffold.

promoting the formation of a type II polyproline helix. Three nonproline residues, often arginine or leucine, also interact with the SH3 domain and appear to confer ligand specificity. Several ligands that were selected from the peptide libraries on the basis of their ability to bind to a fluorescein-conjugated p85a SH3 domain contained the consensus sequence RXLPPRP, where X represents any amino acid except a cysteine residue. In p85a, the sequence KPRPPRPLPVAP in the PI motif was proposed as a likely ligand for p85aSH3, as it contains two partially overlapping motifs (KPRPPRP and RPLPVAP), both of which fulfill the consensus sequence criteria for this domain

(Yu et al., 1994)). In addition, it has been reported that the proline-rich motifs in PI3K p85 can bind to the SH3 domains of Abl, Lck, Fyn, and p85, with the p85 SH3 domain exhibiting the strongest affinity (Kapeller et ai, 1994).

Unlocking the closed conformation of p85a, in which the SH3 domain is bound to its own PI, would free the SH3 domain and allow it to bind dynamin or other exogenous proline-rich ligands. Secondly, unlocking the p85aSH3Pl interaction would allow access to the p85aPl motif by the SH3 domains of the Src family tyrosine kinases. The locking and unlocking of this interaction may be a regulatory mechanism for PI3K, however this will require further investigation. There is, however, evidence that this may be the case, as it has been reported that the proline-rich region in p85 mediates the activation of hpid kinase activity by binding to the Lyn or Fyn SH3 domains (Pleiman

et al., 1994). This type of regulatory mechanism is analogous to that reported for the Src family tyrosine kinases, in which phosphorylation of a carboxy-terminal tyrosine residue regulates kinase activity and substrate binding through co-operative intramolecular binding of the tyrosine phosphorylated tail and the SH2 domain, and a pseudo type II polyproline helix and the SH3 domain (Cooper and Kashishian, 1993; Courtneidge, 1985; Xu et al., 1997), (section 1.3.3.1.3). A similar mechanism has also been shown to be important for the regulation of the Tec family of intracellular tyrosine kinases. The SH3 domain of Itk interacts with an adjacent proline-rich region, and therefore restricts access of the SH3 domain to potential exogenous binding partners (Andreotti et al., 1997). Phosphorylation of a tyrosine residue within the SH3 domain of Btk, a member of the Tec family was reported to disrupt this intramolecular SH3-proline-rich region interaction, thereby liberating the SH3 domain to engage kinase substrates (Park et al., 1996).

As discussed in chapter 4, no GAP activity has been detected in recombinant p85a or PI3K to date. It is possible that the BH domain of p85 may be a GAP for an as yet unidentified GTPase protein, however, it is also possible that the SH3 domain interactions regulate the GAP activity of the neighbouring BH domain. A specific

p85aSH 3 domain or p 8 5 aP l binding event maybe required to unlock the intramolecular interaction before GAP activity is detectable.

The SH3 domain of p85 may therefore provide a link between receptor binding of PI3K and activation of downstream pathways involving SH3 domain and proline-rich motif containing proteins that regulate cellular functions such as cytoskeletal rearrangements and endocytosis. The interaction of p85a and one such SH3 domain containing protein, c-Src , was examined further.