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Comparative proteomic analysis of compartmentalised Ras signalling.

Hernandez-Valladares M, Prior IA - Sci Rep (2015)

Bottom Line: However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated.Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling.Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.

ABSTRACT
Ras proteins are membrane bound signalling hubs that operate from both the cell surface and endomembrane compartments. However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated. To address this, we have performed a global screen of compartmentalised Ras signalling. We find that whilst ER/Golgi- and endosomal-Ras only generate weak outputs, Ras localised to the mitochondria or Golgi significantly and distinctly influence both the abundance and phosphorylation of a wide range of proteins analysed. Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling. The majority of compartmentalised Ras-specific responses are predicted to influence gene expression, RNA splicing and cell proliferation. Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling.

No MeSH data available.


Related in: MedlinePlus

Organellar Ras and experimental scheme.(A) Fluorescence microscopy images showing subcellular locations of transiently expressed Ras (G12V) mutant constructs in HeLa S3 cells. Anti-PDI1, anti-Grasp55, anti-EEA1 and anti-TOM20 antibodies (red) were used to confirm ER, Golgi, endosomal and mitochondrial location, respectively. (B) Workflow for the peptide and phosphopeptide preparations used in this study. The triplex organisation for a single biological replicate is shown.
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f1: Organellar Ras and experimental scheme.(A) Fluorescence microscopy images showing subcellular locations of transiently expressed Ras (G12V) mutant constructs in HeLa S3 cells. Anti-PDI1, anti-Grasp55, anti-EEA1 and anti-TOM20 antibodies (red) were used to confirm ER, Golgi, endosomal and mitochondrial location, respectively. (B) Workflow for the peptide and phosphopeptide preparations used in this study. The triplex organisation for a single biological replicate is shown.

Mentions: To investigate location-specific Ras signalling, we utilised a series of GFP-tagged constitutively active (G12V) Ras chimeras where the C-terminal Ras membrane targeting and localisation motifs has been replaced by organelle-specific targetting motifs19. As positive controls for normal Ras localisation and function we used NRAS and KRAS (Fig. 1A). KRAS displays a predominant plasma membrane distribution with a small fraction on the ER and cytosol. In contrast, NRAS exhibits a significant endomembrane and Golgi pool in addition to cell surface localisation. ER/Golgi, Golgi and mitochondrial Ras are highly restricted to their target organelles based on morphology and/or co-localisation with organelle markers. Endosomal Ras is the least specifically targeted; however, clear co-localisation with the early endosomal marker EEA1 can be seen whilst the nucleus localisation is likely to be unproductive and therefore silent in our study (Fig. 1A).


Comparative proteomic analysis of compartmentalised Ras signalling.

Hernandez-Valladares M, Prior IA - Sci Rep (2015)

Organellar Ras and experimental scheme.(A) Fluorescence microscopy images showing subcellular locations of transiently expressed Ras (G12V) mutant constructs in HeLa S3 cells. Anti-PDI1, anti-Grasp55, anti-EEA1 and anti-TOM20 antibodies (red) were used to confirm ER, Golgi, endosomal and mitochondrial location, respectively. (B) Workflow for the peptide and phosphopeptide preparations used in this study. The triplex organisation for a single biological replicate is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664896&req=5

f1: Organellar Ras and experimental scheme.(A) Fluorescence microscopy images showing subcellular locations of transiently expressed Ras (G12V) mutant constructs in HeLa S3 cells. Anti-PDI1, anti-Grasp55, anti-EEA1 and anti-TOM20 antibodies (red) were used to confirm ER, Golgi, endosomal and mitochondrial location, respectively. (B) Workflow for the peptide and phosphopeptide preparations used in this study. The triplex organisation for a single biological replicate is shown.
Mentions: To investigate location-specific Ras signalling, we utilised a series of GFP-tagged constitutively active (G12V) Ras chimeras where the C-terminal Ras membrane targeting and localisation motifs has been replaced by organelle-specific targetting motifs19. As positive controls for normal Ras localisation and function we used NRAS and KRAS (Fig. 1A). KRAS displays a predominant plasma membrane distribution with a small fraction on the ER and cytosol. In contrast, NRAS exhibits a significant endomembrane and Golgi pool in addition to cell surface localisation. ER/Golgi, Golgi and mitochondrial Ras are highly restricted to their target organelles based on morphology and/or co-localisation with organelle markers. Endosomal Ras is the least specifically targeted; however, clear co-localisation with the early endosomal marker EEA1 can be seen whilst the nucleus localisation is likely to be unproductive and therefore silent in our study (Fig. 1A).

Bottom Line: However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated.Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling.Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.

ABSTRACT
Ras proteins are membrane bound signalling hubs that operate from both the cell surface and endomembrane compartments. However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated. To address this, we have performed a global screen of compartmentalised Ras signalling. We find that whilst ER/Golgi- and endosomal-Ras only generate weak outputs, Ras localised to the mitochondria or Golgi significantly and distinctly influence both the abundance and phosphorylation of a wide range of proteins analysed. Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling. The majority of compartmentalised Ras-specific responses are predicted to influence gene expression, RNA splicing and cell proliferation. Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling.

No MeSH data available.


Related in: MedlinePlus