Requirement of phosphatidylinositol 3-kinase activity for translocation of exogenous aFGF to the cytosol and nucleus.
Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.
Klingenberg
O
O
Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Wiedocha
A
A
Citores
L
L
Olsnes
S
S
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002867:Chromones; D004167:Diphtheria Toxin; D004791:Enzyme Inhibitors; D016220:Fibroblast Growth Factor 1; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one