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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

Compartmentalised Ras responsive phosphosites include a network of RNA processing proteins.(A) Phosphosites exhibiting ≥1.5-fold responsiveness versus the GFP control were shortlisted and GO analysis indicated that proteins associated with DNA and RNA processing and significantly enriched. (B) All of the phosphosites associated with RNA processing proteins are shown; hierarchical clustering indicates similar responses from Golgi, Mito, KRAS and NRAS. Kinase responses from the proteome and phosphoproteome data clustered by cell function ontology. (C) STRING analysis of the RNA processing subset reveals an interactome associated with spliceosome function.
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f5: Compartmentalised Ras responsive phosphosites include a network of RNA processing proteins.(A) Phosphosites exhibiting ≥1.5-fold responsiveness versus the GFP control were shortlisted and GO analysis indicated that proteins associated with DNA and RNA processing and significantly enriched. (B) All of the phosphosites associated with RNA processing proteins are shown; hierarchical clustering indicates similar responses from Golgi, Mito, KRAS and NRAS. Kinase responses from the proteome and phosphoproteome data clustered by cell function ontology. (C) STRING analysis of the RNA processing subset reveals an interactome associated with spliceosome function.

Mentions: An alternative method of collating organelle-specific Ras responses was based on identifying those that substantially differed from the GFP control using an arbitrary 1.5-fold change cut-off. In total we identified 266 responsive phosphopeptides with 215 distinct sites (~80%) being ≥1.5-fold responsive versus the GFP control in at least one of the organellar Ras variants (Supplementary Table 2). GO analysis of the phosphosites exhibiting ≥1.5-fold changes vs GFP indicated that DNA and RNA processing and organisation were major phenotypic targets (Fig. 5A). An illustrative example is depicted using the RNA processing subset of responsive phosphosites (Fig. 5B). The majority of the proteins identified in this subset represent a functional interactome involved in pre-mRNA splice site identification, spliceosome assembly and regulation of RNA splicing (Fig. 5C).


Comparative proteomic analysis of compartmentalised Ras signalling.

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

Compartmentalised Ras responsive phosphosites include a network of RNA processing proteins.(A) Phosphosites exhibiting ≥1.5-fold responsiveness versus the GFP control were shortlisted and GO analysis indicated that proteins associated with DNA and RNA processing and significantly enriched. (B) All of the phosphosites associated with RNA processing proteins are shown; hierarchical clustering indicates similar responses from Golgi, Mito, KRAS and NRAS. Kinase responses from the proteome and phosphoproteome data clustered by cell function ontology. (C) STRING analysis of the RNA processing subset reveals an interactome associated with spliceosome function.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Compartmentalised Ras responsive phosphosites include a network of RNA processing proteins.(A) Phosphosites exhibiting ≥1.5-fold responsiveness versus the GFP control were shortlisted and GO analysis indicated that proteins associated with DNA and RNA processing and significantly enriched. (B) All of the phosphosites associated with RNA processing proteins are shown; hierarchical clustering indicates similar responses from Golgi, Mito, KRAS and NRAS. Kinase responses from the proteome and phosphoproteome data clustered by cell function ontology. (C) STRING analysis of the RNA processing subset reveals an interactome associated with spliceosome function.
Mentions: An alternative method of collating organelle-specific Ras responses was based on identifying those that substantially differed from the GFP control using an arbitrary 1.5-fold change cut-off. In total we identified 266 responsive phosphopeptides with 215 distinct sites (~80%) being ≥1.5-fold responsive versus the GFP control in at least one of the organellar Ras variants (Supplementary Table 2). GO analysis of the phosphosites exhibiting ≥1.5-fold changes vs GFP indicated that DNA and RNA processing and organisation were major phenotypic targets (Fig. 5A). An illustrative example is depicted using the RNA processing subset of responsive phosphosites (Fig. 5B). The majority of the proteins identified in this subset represent a functional interactome involved in pre-mRNA splice site identification, spliceosome assembly and regulation of RNA splicing (Fig. 5C).

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