Role of Src-family kinases in formation and trafficking of macropinosomes.
Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.
Kasahara
Kousuke
K
Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Nakayama
Yuji
Y
Sato
Izumi
I
Ikeda
Kikuko
K
Hoshino
Masaki
M
Endo
Takeshi
T
Yamaguchi
Naoto
N
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019308:Palmitic Acid / Q000378:metabolism; D010873:Pinocytosis / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D060149:Tetraspanin 30; D013997:Time Factors; D019061:src-Family Kinases / Q000378:metabolism*
D015703:Antigens, CD; C554586:CD63 protein, human; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D060149:Tetraspanin 30; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D004815:Epidermal Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases