EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras-MAP kinase pathway.
Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic. Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI. The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented. Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF. Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET. Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane. The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane. We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms.
Larijani
Banafshé
B
Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, WC2A 3PX, London, United Kingdom.
Allen-Baume
Victoria
V
Morgan
Clive P
CP
Li
Michelle
M
Cockcroft
Shamshad
S
D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D002070:Butadienes / Q000494:pharmacology; D019556:COS Cells / Q000187:drug effects; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology; D009930:Organic Chemicals; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018631:ras Proteins / Q000187:drug effects / Q000378:metabolism*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002070:Butadienes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008055:Lipids; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009570:Nitriles; D009930:Organic Chemicals; C466090:PD 98058; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D049294:Phospholipid Transfer Proteins; C113580:U 0126; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D018631:ras Proteins