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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

Compartment-specific Ras network responses.Proteome and phosphoproteome cross-correlations among six Ras (G12V) distinct organellar locations using either GFP or NRAS (G12V) in the denominator of log2-transformed intensity ratios. High correlation values (near 1.0) were red-coloured and clustered together. The influence of changes in protein abundance in the proteome on the their cognate phosphosite responses was removed in the final cross-correlation matrix (Phospho-proteome/Proteome) to reveal high correlation between KRAS, Golgi-Ras and mito-Ras phosphosite responses. Analysis was performed on ratios from MaxQuant analysis of n = 3 biological repeats.
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f3: Compartment-specific Ras network responses.Proteome and phosphoproteome cross-correlations among six Ras (G12V) distinct organellar locations using either GFP or NRAS (G12V) in the denominator of log2-transformed intensity ratios. High correlation values (near 1.0) were red-coloured and clustered together. The influence of changes in protein abundance in the proteome on the their cognate phosphosite responses was removed in the final cross-correlation matrix (Phospho-proteome/Proteome) to reveal high correlation between KRAS, Golgi-Ras and mito-Ras phosphosite responses. Analysis was performed on ratios from MaxQuant analysis of n = 3 biological repeats.

Mentions: To provide a comprehensive overview of organelle response variability we performed cross-correlation and hierarchical clustering with protein and phosphosite ratios using GFP values in the denominator (Fig. 3). Two distinct groups were observed: one that involves Golgi-, Mito-, K- and N- (G12V) Ras and another that involves ER/Golgi- and Endo- (G12V) Ras. Removing the influence of the protein abundance changes within the proteome strengthened the correlation between KRAS, Golgi-Ras and mito-Ras in the phosphosite dataset. When SILAC ratios were calculated using NRAS as the denominator, both Endo and ER-Golgi Ras outputs correlate with GFP (Fig. 3). The similarity to the negative control indicates that these two locations do not support robust Ras signalling. In contrast, the Golgi and mitochondria sustain distinctive responses that do not correlate with the GFP negative control. In this respect they resemble the positive controls of NRAS and especially KRAS, suggesting that these two organelles are capable of supporting significant Ras signalling outputs. These data reveal that each location generates a distinct and network response; that KRAS, NRAS, Golgi-Ras and mito-Ras outputs are robust and broadly similar and that endo-Ras and ER/Golgi-Ras are weak activators of the Ras network.


Comparative proteomic analysis of compartmentalised Ras signalling.

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

Compartment-specific Ras network responses.Proteome and phosphoproteome cross-correlations among six Ras (G12V) distinct organellar locations using either GFP or NRAS (G12V) in the denominator of log2-transformed intensity ratios. High correlation values (near 1.0) were red-coloured and clustered together. The influence of changes in protein abundance in the proteome on the their cognate phosphosite responses was removed in the final cross-correlation matrix (Phospho-proteome/Proteome) to reveal high correlation between KRAS, Golgi-Ras and mito-Ras phosphosite responses. Analysis was performed on ratios from MaxQuant analysis of n = 3 biological repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Compartment-specific Ras network responses.Proteome and phosphoproteome cross-correlations among six Ras (G12V) distinct organellar locations using either GFP or NRAS (G12V) in the denominator of log2-transformed intensity ratios. High correlation values (near 1.0) were red-coloured and clustered together. The influence of changes in protein abundance in the proteome on the their cognate phosphosite responses was removed in the final cross-correlation matrix (Phospho-proteome/Proteome) to reveal high correlation between KRAS, Golgi-Ras and mito-Ras phosphosite responses. Analysis was performed on ratios from MaxQuant analysis of n = 3 biological repeats.
Mentions: To provide a comprehensive overview of organelle response variability we performed cross-correlation and hierarchical clustering with protein and phosphosite ratios using GFP values in the denominator (Fig. 3). Two distinct groups were observed: one that involves Golgi-, Mito-, K- and N- (G12V) Ras and another that involves ER/Golgi- and Endo- (G12V) Ras. Removing the influence of the protein abundance changes within the proteome strengthened the correlation between KRAS, Golgi-Ras and mito-Ras in the phosphosite dataset. When SILAC ratios were calculated using NRAS as the denominator, both Endo and ER-Golgi Ras outputs correlate with GFP (Fig. 3). The similarity to the negative control indicates that these two locations do not support robust Ras signalling. In contrast, the Golgi and mitochondria sustain distinctive responses that do not correlate with the GFP negative control. In this respect they resemble the positive controls of NRAS and especially KRAS, suggesting that these two organelles are capable of supporting significant Ras signalling outputs. These data reveal that each location generates a distinct and network response; that KRAS, NRAS, Golgi-Ras and mito-Ras outputs are robust and broadly similar and that endo-Ras and ER/Golgi-Ras are weak activators of the Ras network.

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