The N-terminus of phosphoinositide 3-kinase-C2beta regulates lipid kinase activity and binding to clathrin.
The class II phosphoinositide 3-kinase (PI3K)-C2beta 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-C2beta 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-C2beta following stimulation with EGF. Deletion of the first proline rich region within the N-terminus precluded recruitment of PI3K-C2beta 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-C2beta-GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K-C2beta with clathrin was confirmed by co-immunoprecipitation from cell lysates while intracellular co-localisation of PI3K-C2beta and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K-C2beta isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport.
Wheeler
Matthew
M
Faculty of Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom.
Domin
Jan
J
D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D058541:Class II Phosphatidylinositol 3-Kinases; D002966:Clathrin / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006801:Humans; D008055:Lipids; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D010770:Phosphotransferases; D014407:Tumor Cells, Cultured
D002966:Clathrin; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008055:Lipids; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D058541:Class II Phosphatidylinositol 3-Kinases; C502616:PIK3C2B protein, human; D066246:ErbB Receptors