Differential subcellular membrane recruitment of Src may specify its downstream signalling
Philippe de Diesbach1, Thierry Medts, Sarah Carpentier
1Université catholique de Louvain and de Duve Institute (ICP), CELL Unit, UCL-7541, avenue Hippocrate 75, B-1200 Brussels, Belgium.
Abstract:
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 degrees 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 ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) 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 MbetaCD, 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.
Insights
Activated Src kinase is recruited to distinct membrane compartments, influencing cell signaling pathways. This localization impacts both MAP-kinase and PI3-kinase pathways, with implications for cancer research.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Most Src family kinases associate with membrane "lipid rafts" to coordinate signaling.
- The localization of monoacylated Src, often hyperactive in carcinomas, to "lipid rafts" is debated.
Purpose of the Study:
- Investigate how Src tyrosine-kinase activation affects its membrane recruitment, subcellular localization, and signaling.
- Determine Src localization dynamics in response to activation in polarized cells.
Main Methods:
- Utilized polarized Madin-Darby canine kidney (MDCK) cells expressing a thermosensitive v-Src variant (tsLA31).
- Assessed Src membrane recruitment and "lipid raft" association via detergent resistance and sucrose density gradient flotation.
- Examined subcellular localization using immunofluorescence and analyzed signaling pathway activation (MAP-kinase, PI3-kinase).
- Investigated the role of cholesterol in Src localization and signaling using methyl-beta-cyclodextrin (MbetaCD).
Main Results:
- Src kinase thermoactivation correlated with recruitment to sedimentable membranes, resisting detergent solubilization and associating with "lipid rafts".
- Activated Src localized to apical endosomes/macropinosomes and the apical plasma membrane, with the latter overlapping GM1 but not caveolin-1/flotillin-2.
- Cholesterol depletion by MbetaCD reduced Src association with "rafts" and abolished apical plasma membrane localization.
- Src activation sequentially activated MAP-kinase (ERK1/2) and PI3-kinase (Akt) pathways.
- MAP-kinase activation was MbetaCD-insensitive, while Akt phosphorylation and Src-induced apical endocytosis were suppressed by MbetaCD.
Conclusions:
- Activated Src localizes to distinct membrane compartments, enabling differential signaling.
- Signaling via ERK1/2 occurs at non-raft domains on endosomes, while PI3-kinase-Akt signaling occurs on specific rafts at the apical plasma membrane.
- This model of Src localization and signaling warrants further investigation for its applicability to c-Src in carcinomas.
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