Abstract
The class II phosphoinositide 3‐kinase (PI3K)‐C2β is recruited to polypeptide growth factor receptors following ligand stimulation. In contrast to the class I A p85/p110 heterodimers, this interaction is dependent upon proline residues present within the N‐terminal sequence of the 3‐phosphoinositide kinase. However, the mechanism by which PI3K‐C2β catalytic activity is regulated currently remains unknown. In many tumours, increased expression of ErbB receptors confers a poor prognosis. We demonstrate that increased expression of EGFR enhanced its association with PI3K‐C2β following stimulation with EGF. Deletion of the first proline rich region within the N‐terminus precluded recruitment of PI3K‐C2β to activated EGFR. Although deletion of the first proline rich motif also rendered the enzyme catalytically inactive, further deletions (residues 1–148 and 1–261) that removed the second and third proline rich motifs increased kinase activity. These data confirm a regulatory role for the N‐terminus of class II PI3K enzymes suggesting that catalytic activity is regulated by factors that associate with this region during recruitment to activated growth factor receptors. Using an N‐terminal PI3K‐C2β‐GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K‐C2β with clathrin was confirmed by co‐immunoprecipitation from cell lysates while intracellular co‐localisation of PI3K‐C2β and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K‐C2β isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport. © 2005 Wiley‐Liss, Inc.