Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization Journal Article
| Authors: | Chen, X.; Resh, M. D. |
| Article Title: | Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization |
| Abstract: | Binding of proteins to the plasma membrane can be achieved with various membrane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of different membrane targeting motifs influences the signaling capacity of membrane-bound signal transduction proteins by directing the proteins to different membrane microdomains. We used c-Raf-1 as a model for a signaling protein that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn, and Src proteins were fused to the N or C terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to nearly equivalent levels. The extent of localization of the Raf-1 constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting drug methyl-β-cyclodextrin (MβCD) caused activation of MAPK to levels equivalent to those achieved with membrane-targeted Raf constructs. The use of pharmacological agents as well as dominant negative mutants revealed that MAPK activation by MβCD proceeds via a phosphoinositide 3-kinase-dependent mechanism that is Ras/Raf-independent. We conclude that cholesterol depletion from the plasma membrane by MβCD constitutes an alternative pathway for activating MAPK. |
| Keywords: | signal transduction; mitogen activated protein kinase; unclassified drug; raf protein; nonhuman; animal cell; animal; metabolism; animals; mitogen activated protein kinase inhibitor; carboxy terminal sequence; protein targeting; protein; enzyme activation; protein serine threonine kinase; cos cells; phosphatidylinositol 3 kinase; physiology; animalia; chemistry; amino terminal sequence; protein-serine-threonine kinases; cell membrane; 1-phosphatidylinositol 3-kinase; cell membranes; cell strain cos1; ras protein; cholesterol; fatty acids; k ras protein; mitogen-activated protein kinases; mutagenesis; enzymes; membrane binding; proto-oncogene proteins c-raf; rabbits; beta cyclodextrin derivative; methyl beta cyclodextrin; methyl-beta-cyclodextrin; beta-cyclodextrins; protein kinase fyn; drug products; membrane microdomains; rabbit; cyclodextrins; cyclodextrin; priority journal; article; plasma membranes; protein c raf 1; protein src; membrane raft |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 37 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-09-14 |
| Start Page: | 34617 |
| End Page: | 34623 |
| Language: | English |
| DOI: | 10.1074/jbc.M103995200 |
| PUBMED: | 11457834 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
