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.
Experimental Cell Research
|March 5, 2008
Summary
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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