TY - JOUR
T1 - Differential subcellular membrane recruitment of Src may specify its downstream signalling
AU - de Diesbach, Philippe
AU - Medts, Thierry
AU - Carpentier, Sarah
AU - D'Auria, Ludovic
AU - Van Der Smissen, Patrick
AU - Platek, Anna
AU - Mettlen, Marcel
AU - Caplanusi, Adrian
AU - van den Hove, Marie France
AU - Tyteca, Donatienne
AU - Courtoy, Pierre J.
N1 - Funding Information:
We gratefully thank Drs. M. Mareel and J. Behrens for providing the MDCK/ ts LA31 cell line, Dr. G. Ojakian for the kind gift of MAb to gp135/podocalyxin, Dr. M. Rider for carefully revising the manuscript, and Mrs. L. Thanh and M. Leruth and Mr. Y. Marchand for their excellent technical, pictorial and editorial assistance. This work was supported by grants from UCL, FNRS, Région wallonne, Région bruxelloise, ARC and IUAP (Belgium) and EuReGene. PdD and DT are Postdoctoral Researchers at the F.R.S-FNRS, at which LDA is Research Fellow; TM is supported by a Televie PhD grant.
PY - 2008/4/15
Y1 - 2008/4/15
N2 - Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 °C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.
AB - Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 °C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.
KW - Endosomes
KW - MAP-kinase
KW - PI3-kinase
KW - Plasma membrane
KW - Rafts
KW - Src
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U2 - 10.1016/j.yexcr.2008.01.015
DO - 10.1016/j.yexcr.2008.01.015
M3 - Article
C2 - 18316074
AN - SCOPUS:40949135021
SN - 0014-4827
VL - 314
SP - 1465
EP - 1479
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 7
ER -