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p70S6K is regulated by focal adhesion kinase and is required for Src-selective autophagy.

Sandilands E, Schoenherr C, Frame MC - Cell. Signal. (2015)

Bottom Line: Specifically, in SCCs that are genetically deficient for FAK, there is reduced phosphorylation of Akt, p70S6K and S6, and signalling to Akt-p70S6K-S6 is more sensitive to inhibition by multiple agents that suppress the pathway.This is associated with loss of a complex between p-Src and the autophagy protein LC3, a biochemical surrogate of impaired Src-selective autophagy.We therefore deduce that the FAK-regulated signalling module PDK1-Akt-p70S6K that controls Src's intracellular trafficking operates at Src-containing autophagosomes.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Signalling proteins localise to Src-containing autophagosomes. FAK −/− cells were fixed and stained with (A) anti-p-PDK1 S241 (upper panels), anti-p-Akt S473 (lower panels) or (B) anti-p-p70S6K T389 (upper panels), anti-mTOR (lower panels) and with anti-p-Src Y416 and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate co-localisation while broken arrows show its absence. Scale bars: 20 μm. (C) Amount of co-localisation per cell was calculated by measuring by the distance between the intensity peaks of the different fluorescent signals. Results are presented as mean ± s.d. (n = 20 intensity peaks from 5 cells). (D) Src was immunoprecipitated from SCC cells and immunoblotting performed with anti-p70S6K and anti-Src. (E) Model of signalling at Src positive autophagosomes. Schematic depicting how the use of inhibitors influences the trafficking of active Src to autophagic puncta in the absence of FAK.
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f0020: Signalling proteins localise to Src-containing autophagosomes. FAK −/− cells were fixed and stained with (A) anti-p-PDK1 S241 (upper panels), anti-p-Akt S473 (lower panels) or (B) anti-p-p70S6K T389 (upper panels), anti-mTOR (lower panels) and with anti-p-Src Y416 and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate co-localisation while broken arrows show its absence. Scale bars: 20 μm. (C) Amount of co-localisation per cell was calculated by measuring by the distance between the intensity peaks of the different fluorescent signals. Results are presented as mean ± s.d. (n = 20 intensity peaks from 5 cells). (D) Src was immunoprecipitated from SCC cells and immunoblotting performed with anti-p70S6K and anti-Src. (E) Model of signalling at Src positive autophagosomes. Schematic depicting how the use of inhibitors influences the trafficking of active Src to autophagic puncta in the absence of FAK.

Mentions: We next examined the intracellular localisation of activated components of the PDK1/Akt/p70S6K signalling pathway. We found that all of these co-localised to autophagic puncta with active Src (Fig. 4A and B respectively, solid arrows in merged images). Although we observed mTOR in cytoplasmic puncta, it did not visibly co-localise with p-Src-containing puncta in FAK-deficient cells (Fig. 2B, lower panels, broken arrow in merged image). A measure of the co-localisation (or lack thereof in the case of mTOR) is presented as distance between intensity peaks of co-stained proteins (Fig. 2C). We also identified a biochemical complex that contained both Src and p70S6K, although we have no evidence that this complex is via direct binding (Fig. 4D). These data imply that the intracellular puncta that contain autophagy proteins also physically serve as a sub-locale for the activated kinase components of the signalling pathway – PDK1/Akt/p70S6K – which promotes efficient targeting of Src to these structures when Src cannot be tethered at focal adhesion complexes in the absence of FAK.


p70S6K is regulated by focal adhesion kinase and is required for Src-selective autophagy.

Sandilands E, Schoenherr C, Frame MC - Cell. Signal. (2015)

Signalling proteins localise to Src-containing autophagosomes. FAK −/− cells were fixed and stained with (A) anti-p-PDK1 S241 (upper panels), anti-p-Akt S473 (lower panels) or (B) anti-p-p70S6K T389 (upper panels), anti-mTOR (lower panels) and with anti-p-Src Y416 and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate co-localisation while broken arrows show its absence. Scale bars: 20 μm. (C) Amount of co-localisation per cell was calculated by measuring by the distance between the intensity peaks of the different fluorescent signals. Results are presented as mean ± s.d. (n = 20 intensity peaks from 5 cells). (D) Src was immunoprecipitated from SCC cells and immunoblotting performed with anti-p70S6K and anti-Src. (E) Model of signalling at Src positive autophagosomes. Schematic depicting how the use of inhibitors influences the trafficking of active Src to autophagic puncta in the absence of FAK.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508348&req=5

f0020: Signalling proteins localise to Src-containing autophagosomes. FAK −/− cells were fixed and stained with (A) anti-p-PDK1 S241 (upper panels), anti-p-Akt S473 (lower panels) or (B) anti-p-p70S6K T389 (upper panels), anti-mTOR (lower panels) and with anti-p-Src Y416 and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate co-localisation while broken arrows show its absence. Scale bars: 20 μm. (C) Amount of co-localisation per cell was calculated by measuring by the distance between the intensity peaks of the different fluorescent signals. Results are presented as mean ± s.d. (n = 20 intensity peaks from 5 cells). (D) Src was immunoprecipitated from SCC cells and immunoblotting performed with anti-p70S6K and anti-Src. (E) Model of signalling at Src positive autophagosomes. Schematic depicting how the use of inhibitors influences the trafficking of active Src to autophagic puncta in the absence of FAK.
Mentions: We next examined the intracellular localisation of activated components of the PDK1/Akt/p70S6K signalling pathway. We found that all of these co-localised to autophagic puncta with active Src (Fig. 4A and B respectively, solid arrows in merged images). Although we observed mTOR in cytoplasmic puncta, it did not visibly co-localise with p-Src-containing puncta in FAK-deficient cells (Fig. 2B, lower panels, broken arrow in merged image). A measure of the co-localisation (or lack thereof in the case of mTOR) is presented as distance between intensity peaks of co-stained proteins (Fig. 2C). We also identified a biochemical complex that contained both Src and p70S6K, although we have no evidence that this complex is via direct binding (Fig. 4D). These data imply that the intracellular puncta that contain autophagy proteins also physically serve as a sub-locale for the activated kinase components of the signalling pathway – PDK1/Akt/p70S6K – which promotes efficient targeting of Src to these structures when Src cannot be tethered at focal adhesion complexes in the absence of FAK.

Bottom Line: Specifically, in SCCs that are genetically deficient for FAK, there is reduced phosphorylation of Akt, p70S6K and S6, and signalling to Akt-p70S6K-S6 is more sensitive to inhibition by multiple agents that suppress the pathway.This is associated with loss of a complex between p-Src and the autophagy protein LC3, a biochemical surrogate of impaired Src-selective autophagy.We therefore deduce that the FAK-regulated signalling module PDK1-Akt-p70S6K that controls Src's intracellular trafficking operates at Src-containing autophagosomes.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, United Kingdom.

No MeSH data available.


Related in: MedlinePlus