Intracellular Movement of Green Fluorescent Protein–tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling

Correspondence to: William J. Gullick, Receptor Biology Laboratory, Imperial Cancer Research Fund Molecular Oncology Unit, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. Tel:44-181-383-4322 Fax:44-181-383-3258 E-mail:gullick{at}icrf.icnet.uk.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH₂-terminal part of the protein as well as its SH2 domains.

Key Words: phosphatidylinositol 3-kinase, green fluorescent protein, erbB-3, focal complexes, tyrosine kinase

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