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Proteomic analysis of endosomes from genetically modified p14/MP1 mouse embryonic fibroblasts
Taras Stasyk1, Johann Holzmann, Sonja Stumberger
1Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Abstract:
The p14/MP1 scaffold complex binds MEK1 and ERK1/2 on late endosomes, thus regulating the strength, duration and intracellular location of MAPK signaling. By organelle proteomics we have compared the protein composition of endosomes purified from genetically modified p14⁻/⁻, p14+/⁻ and p14(rev) mouse embryonic fibroblasts. The latter ones were reconstituted retrovirally from p14⁻/⁻ mouse embryonic fibroblasts by reexpression of pEGFP-p14 at equimolar ratios with its physiological binding partner MP1, as shown here by absolute quantification of MP1 and p14 proteins on endosomes by quantitative MS using the Equimolarity through Equalizer Peptide strategy. A combination of subcellular fractionation, 2-D DIGE and MALDI-TOF/TOF MS revealed 31 proteins differentially regulated in p14⁻/⁻ organelles, which were rescued by reexpression of pEGFP-p14 in p14⁻/⁻ endosomes. Regulated proteins are known to be involved in actin remodeling, endosomal signal transduction and trafficking. Identified proteins and their in silico interaction networks suggested that endosomal signaling might regulate such major cellular functions such as proliferation, differentiation, migration and survival.
Insights
The p14/MP1 scaffold complex regulates mitogen-activated protein kinase (MAPK) signaling at endosomes. Loss of p14 protein alters endosomal protein composition, impacting cell functions like proliferation and migration.
Area of Science:
- Cell Biology
- Molecular Biology
- Proteomics
Background:
- The p14/MP1 scaffold complex is crucial for regulating mitogen-activated protein kinase (MAPK) signaling.
- This complex localizes to late endosomes, influencing signal strength, duration, and location.
- Understanding the p14/MP1 complex's role in endosomal protein composition is key to deciphering MAPK pathway regulation.
Purpose of the Study:
- To investigate the impact of p14 protein deficiency on the proteomic landscape of endosomes.
- To identify proteins regulated by the p14/MP1 complex on endosomes.
- To explore the functional consequences of altered endosomal protein composition on cellular processes.
Main Methods:
- Organelle proteomics using subcellular fractionation, 2-D DIGE, and MALDI-TOF/TOF MS.
- Comparative analysis of endosomal protein composition in p14 knockout (p14⁻/⁻), heterozygous (p14⁺/⁻), and reconstituted (p14(rev)) mouse embryonic fibroblasts.
- Quantitative mass spectrometry (MS) employing the Equimolarity through Equalizer Peptide strategy for absolute protein quantification.
Main Results:
- 31 proteins were found to be differentially regulated in p14⁻/⁻ endosomes compared to wild-type.
- Re-expression of p14 protein in p14⁻/⁻ cells rescued the observed changes in endosomal protein composition.
- The identified differentially regulated proteins are involved in actin remodeling, endosomal signal transduction, and trafficking.
Conclusions:
- The p14/MP1 complex significantly shapes the endosomal proteome.
- Alterations in endosomal protein composition due to p14 deficiency impact critical cellular functions, including proliferation, differentiation, migration, and survival.
- In silico analysis of identified proteins and their networks suggests a role for endosomal signaling in regulating major cellular processes.
