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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

Phosphosites displaying organelle-specific responses.(A) Ratios exhibiting changes in abundance were subjected to unsupervised clustering with the Fuzzy c means algorithm using GProX. Clusters corresponding to ten different response patterns were identified. The number (n) in each cluster is indicated. (B) GO analysis indicates that proteins associated with RNA processing, gene expression, the cytoskeleton and cell proliferation are significantly enriched. (C) Members of cluster 1, enriched for RNA processing proteins are highlighted in a representative KRAS versus ER/Golgi-Ras scatter graph.
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f4: Phosphosites displaying organelle-specific responses.(A) Ratios exhibiting changes in abundance were subjected to unsupervised clustering with the Fuzzy c means algorithm using GProX. Clusters corresponding to ten different response patterns were identified. The number (n) in each cluster is indicated. (B) GO analysis indicates that proteins associated with RNA processing, gene expression, the cytoskeleton and cell proliferation are significantly enriched. (C) Members of cluster 1, enriched for RNA processing proteins are highlighted in a representative KRAS versus ER/Golgi-Ras scatter graph.

Mentions: In order to identify the responses associated with each location, we subjected the datasets to unsupervised fuzzy c-means clustering analysis using GProX23. This identifies genes that exhibit matching responses across the phosphoproteome (Fig. 4) or proteome datasets (Supplementary Figure 2 and Supplementary Table 1). Intriguingly, only KRAS exhibits a uniquely coupled subset of responses (Fig. 4A, cluster 1). Gene ontology (GO) analysis reveals a significant enrichment for genes associated with DNA modification, transcription and RNA splicing within the KRAS subset (Fig. 4B). A representative comparison with ER/Golgi-Ras illustrates the reduced relative phosphorylation in cells harbouring KRAS G12V (Fig. 4C). Amongst the KRAS-specific responders is the transcription factor E2F1 that plays an important role in regulating the expression of genes required for DNA replication and cell cycle progression24. The most pronounced KRAS-specific response is seen with Ser20 of the transcriptional repressor and p120Ras GAP associated protein KHDRBS1 (SAM68)25. Although this site has no described regulatory function, it is responsive to EGF stimulation26.


Comparative proteomic analysis of compartmentalised Ras signalling.

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

Phosphosites displaying organelle-specific responses.(A) Ratios exhibiting changes in abundance were subjected to unsupervised clustering with the Fuzzy c means algorithm using GProX. Clusters corresponding to ten different response patterns were identified. The number (n) in each cluster is indicated. (B) GO analysis indicates that proteins associated with RNA processing, gene expression, the cytoskeleton and cell proliferation are significantly enriched. (C) Members of cluster 1, enriched for RNA processing proteins are highlighted in a representative KRAS versus ER/Golgi-Ras scatter graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Phosphosites displaying organelle-specific responses.(A) Ratios exhibiting changes in abundance were subjected to unsupervised clustering with the Fuzzy c means algorithm using GProX. Clusters corresponding to ten different response patterns were identified. The number (n) in each cluster is indicated. (B) GO analysis indicates that proteins associated with RNA processing, gene expression, the cytoskeleton and cell proliferation are significantly enriched. (C) Members of cluster 1, enriched for RNA processing proteins are highlighted in a representative KRAS versus ER/Golgi-Ras scatter graph.
Mentions: In order to identify the responses associated with each location, we subjected the datasets to unsupervised fuzzy c-means clustering analysis using GProX23. This identifies genes that exhibit matching responses across the phosphoproteome (Fig. 4) or proteome datasets (Supplementary Figure 2 and Supplementary Table 1). Intriguingly, only KRAS exhibits a uniquely coupled subset of responses (Fig. 4A, cluster 1). Gene ontology (GO) analysis reveals a significant enrichment for genes associated with DNA modification, transcription and RNA splicing within the KRAS subset (Fig. 4B). A representative comparison with ER/Golgi-Ras illustrates the reduced relative phosphorylation in cells harbouring KRAS G12V (Fig. 4C). Amongst the KRAS-specific responders is the transcription factor E2F1 that plays an important role in regulating the expression of genes required for DNA replication and cell cycle progression24. The most pronounced KRAS-specific response is seen with Ser20 of the transcriptional repressor and p120Ras GAP associated protein KHDRBS1 (SAM68)25. Although this site has no described regulatory function, it is responsive to EGF stimulation26.

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