In this study we sought to delineate the binding site on PI3K-C2β involved in recruiting this enzyme to the activated EGFR (2
). Although the N terminus of this PI3K enzyme mediates this association, it lacks any domain that can directly bind the receptor. We have produced three independent pieces of data to demonstrate the involvement of its proline-rich motifs in this effect. Figure shows that the N-terminal fragment of PI3K-C2β but not PI3K-C2α affinity purified activated EGFR from cell lysates. The N terminus of PI3K-C2α (13
) and PI3K-C2β both contain motifs that form the P-X-X-P consensus that favors interaction with SH3 domains (20
). Secondly, peptides based on each proline-rich region were able to competitively attenuate the interaction between PI3K-C2β and EGFR (Fig. ). In contrast, a control peptide based on an adjacent sequence had no effect. This approach was successfully used in many previous studies to define the sites of protein-protein interaction (3
). Finally, fragments of the PI3K-C2β N terminus expressed as GST fusions demonstrated that either two proline-rich motifs are required for EGFR binding or the proline-rich motif at residues 144 to 149 is critical for this interaction (Fig. ). However, data presented in Fig. show that peptides derived from all three proline-rich motifs are equally able to displace EGFR binding, making this latter possibility less likely.
Given the importance of the proline-rich motifs in PI3K-C2β–EGFR association we sought to identify a molecule that could mediate this interaction. From our earliest experiments we recognized that both the Shc and Grb2 adaptor proteins were isolated by the PI3K-C2β N terminus together with EGFR from cell lysates (Fig. and ). Of these two molecules, only Grb2 contains an SH3 domain capable of binding proline-rich motifs. The Grb2 adaptor (also termed Ash) is the human homologue of the Caenorhabditis elegans
protein Sem-5 and consists of a single SH2 domain flanked by two SH3 domains. Recruitment of Grb2 to activated EGFR is mediated by the SH2 domain and can occur either directly or via phosphorylated Shc (4
). The degree to which Grb2 associates directly with the EGFR or indirectly via Shc appears to be dependent upon the cell type examined, although direct interaction with the receptor is often favored (36
). Each SH3 domain preferentially binds proteins containing proline-rich motifs that adopt a left-handed polyproline type II helix with the minimal consensus sequence P-X-X-P (33
). However, structural and binding studies have since refined this consensus to P-X-X-P-X-R (6
). Each of the three proline-rich motifs present within the N terminus of PI3K-C2β obeys this consensus. The best-characterized binding partner of the Grb2 SH3 domain is the nucleotide exchange factor SOS that in turn acts as a positive regulator of p21ras
). Many studies have demonstrated that the Grb2 SH3 domains bind effectors in addition to SOS. These include adaptor proteins (Gab 1 and 2, Cbl, and Slp-76) (21
), phosphotyrosine phosphatases (SHP-2, PEST) (10
), serine/threonine kinases (MEKK1) (34
), several proteins implicated in cytoskeletal reorganization (dynamin, N-Wasp) (20
), receptors (CD28) (29
), and other guanine nucleotide exchange factors for ras-related proteins (Vav, C3G) (38
We show that recombinant Grb2 directly binds full-length PI3K-C2β in vitro (Fig. ). Data presented in Fig. demonstrate that both the N-terminal and C-terminal Grb2 SH3 domain can bind PI3K-C2β although, in agreement with Fig. , two proline-rich regions were required. It remains unclear why Grb2 binds PI3K-C2β(2-157) and PI3K-C2β(2-255) with equal affinity yet EGFR binding was significantly greater using fragment 2-255. It suggests that the association of Grb2 and PI3K-C2β occurs independently of the EGFR and that this complex forms first within the cell before translocating to the activated receptor. Although it is possible that residues 158 to 255 of PI3K-C2β contain a motif that stabilizes the PI3K-C2β–EGFR interaction, we have expressed this region as a GST fusion protein and found that it does not bind phosphorylated EGFR independently (data not shown). Constitutive interaction between endogenous Grb2 and PI3K-C2β was also demonstrated when the PI3K enzyme was identified in immunoprecipitates of Grb2 from numerous cell lysates (Fig. and ), including one cell line (HEK293) that was transfected with recombinant PI3K-C2β. Figure shows that while EGF stimulation translocates Grb2 to the activated EGFR, it does not alter the stoichiometry of PI3K-C2β or SOS binding, nor does it display a dramatic effect on the catalytic activity of the PI3K enzyme in situ (Fig. ). In contrast, data presented in Fig. show that the interaction of Grb2 and immunoprecipitated EGFR dramatically increased (sevenfold) PI3K-C2β lipid kinase activity. The effect of EGFR was unexpected, given that the receptor does not directly bind PI3K-C2β. However, since the EGFR was isolated by immunoprecipitation from cell lysates, these preparations might contain associated molecules that exert this effect. The increase in specific activity conferred by EGFR and Grb2 was neither synergistic nor additive, suggesting that they share the same mechanism of activation. It is also unclear why addition of phosphorylated EGFR produced a lesser activation than nonphosphorylated receptor. However, this finding was consistent with data presented in Fig. for A431 and HEK293 cells.
Further studies are required to determine if Grb2 is the only adaptor able to mediate association of PI3K-C2β to EGFR. Other SH3 domain-containing proteins recruited to the activated EGFR may also play a role. Potential candidates include Nck (17
), phospholipase Cγ, ras GTPase-activating protein GAP1(m), and vav-2 (31
). Since our data suggest the need for at least two proline-rich motifs (Fig. and ), it seems unlikely that an adaptor containing a single SH3 domain could mediate the interaction between EGFR and PI3K-C2β without the formation of a higher-order complex. Indeed, despite the presence of an SH3 domain, p85 adaptor protein cannot bind PI3K-C2β (1
). Our previous failure to identify a protein that coimmunoprecipitates with either PI3K-C2α or PI3K-C2β in high stoichiometry contrasts with the association of class IA catalytic subunits and the p85 adaptor (2
). However, those experiments were performed using antisera directed against the N-terminal sequence of the class II PI3K enzymes. Consequently, it is possible that proteins associated with PI3K-C2β could block its antigenic epitopes or that an excess of antibody competes with binding partners for similar or overlapping epitopes, resulting in their dissociation.
Important questions remain regarding the significance of class II PI3K recruitment for biological responses downstream of EGFR. One possibility is their involvement in the regulation of receptor internalization. Although the mechanism of EGFR internalization has attracted particular attention, little is understood about which signals trigger their recruitment into clathrin-coated vesicles. Pharmacological inhibition using wortmannin clearly demonstrates a role for PI3K activity (22
), and we have recently confirmed the presence of PI3K-C2α in clathrin-coated vesicle preparations and its ability to regulate clathrin-mediated endocytosis and sorting in the trans
Golgi network (12
). Ligand binding triggers recruitment of EGFR to clathrin-coated pits and its subsequent delivery to lysosomes for degradation. Experiments using fluorescent GFP-tagged p85α and a chimeric EGFR–ErbB-3 receptor show that despite recruitment of the class I PI3K adaptor and a clustering of staining into patches, there was no evidence of receptor internalization or its association with clathrin-containing endosomes following EGF stimulation (19
). Interestingly, Grb2 is also required for efficient receptor endocytosis (46
). Characterization of the FYVE domain (8
) has illustrated how PI3K-dependent vesicle trafficking is regulated by generation of PtdIns3P rather than by PtdIns(3,4)P2
, which are considered to be the principle products of class IA PI3K enzyme activity. Although production of PtdIns3P has been largely attributed to the class III PI3K vps34p homologue, the class II PI3K enzymes may also contribute to its generation in vivo and therefore play a regulatory role in these events.
Since both PI3K-C2α and PI3K-C2β share an extensive and overlapping tissue distribution and play a role downstream of several receptors, it is important to establish if they are regulated in a similar or disparate manner. Although both class II PI3K isozymes lie downstream of the activated EGFR, the results of this study demonstrate that PI3K-C2β and not PI3K-C2α is recruited to the receptor by a mechanism involving proline-rich regions and the Grb2 adaptor. Consequently, this observation offers scope for the differential regulation of PI3K-C2α and PI3K-C2β catalytic activity and thus the development of compounds to selectively antagonize the action of each class II PI3K isozyme.