Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells.
Kidins220 (kinase D-interacting substrate of 220 kDa) is a novel neurospecific protein recently cloned as the first substrate for the Ser/Thr kinase protein kinase D (PKD). Herein we report that Kidins220 is constitutively associated to lipid rafts in PC12 cells, rat primary cortical neurons, and brain synaptosomes. Immunocytochemistry and confocal microscopy together with sucrose gradient fractionation show co-localization of Kidins220 and lipid raft-associated proteins. In addition, cholesterol depletion of cell membranes with methyl-beta-cyclodextrin dramatically alters Kidins220 localization and detergent solubility. By studying the putative involvement of lipid rafts in PKD activation and signaling we have found that active PKD partitions in lipid raft fractions after sucrose gradient centrifugation and that green fluorescent protein-PKD translocates to lipid raft microdomains at the plasma membrane after phorbol ester treatment. Strikingly, lipid rafts disruption by methyl-beta-cyclodextrin delays green fluorescent protein-PKD translocation, as determined by live cell confocal microscopy, and activates PKD, increasing Kidins220 phosphorylation on Ser(919) by a mechanism involving PKCepsilon and the small soluble tyrosine kinase Src. Collectively, these results reveal the importance of lipid rafts on PKD activation, translocation, and downstream signaling to its substrate Kidins220.
Cabrera-Poch
Noemí
N
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), C/Arturo Duperier 4, 28029-Madrid, Spain.
Sánchez-Ruiloba
Lucía
L
Rodríguez-Martínez
María
M
Iglesias
Teresa
T
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000378:metabolism; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000737:chemistry; D013574:Synaptosomes / Q000378:metabolism; D014162:Transfection; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; C482591:KIDINS220 protein, human; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D049452:Green Fluorescent Proteins; D012694:Serine; D002784:Cholesterol; C089212:protein kinase D; D011493:Protein Kinase C