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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

Kinase inhibitors of Src-selective autophagy. (A) FAK −/− cells were treated with the inhibitors A–F for 24 h. Cells were fixed and stained with anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Scale bars: 20 μm. (B) Percentage of cells with active Src localising to intracellular structures was quantified and data presented as mean ± s.d. Significance for all treatments is p < 0.01 (n = 3). (C) LC3B was immunoprecipitated from FAK −/− cell lysates treated with kinase inhibitors. Then immunoblotting was performed using anti-p-Src Y416 and anti-LC3B antibodies.
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f0010: Kinase inhibitors of Src-selective autophagy. (A) FAK −/− cells were treated with the inhibitors A–F for 24 h. Cells were fixed and stained with anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Scale bars: 20 μm. (B) Percentage of cells with active Src localising to intracellular structures was quantified and data presented as mean ± s.d. Significance for all treatments is p < 0.01 (n = 3). (C) LC3B was immunoprecipitated from FAK −/− cell lysates treated with kinase inhibitors. Then immunoblotting was performed using anti-p-Src Y416 and anti-LC3B antibodies.

Mentions: Next we performed a small drug screen using a commercially available kinase inhibitor library. Adherent FAK −/− cells were treated with 80 different kinase inhibitors (Tocriscreen Kinase Inhibitor Toolbox) for 24 h, then fixed and stained with p-Src Y416 antibody, Deep Red Cell Mask and DAPI, so as to visualise autophagosomes and adhesions, cell cytoplasm and nucleus, respectively. Cells were imaged and analysed using an Olympus Scan-R microscope to calculate the number of Src-positive puncta per cell in untreated FAK-WT (less than 15 puncta per cell) and untreated FAK −/− (58 puncta per cell) cells as negative and positive controls. We focused on 6 chemical entities that significantly reduced the number of Src-positive puncta detected per cell (Fig. 2A and quantified in 2B), but were not in the class of chemicals that would inhibit Src phosphorylation per se (for example, there was no significant decrease in either the amount of total or active Src after treatment of FAK-WT cells with these agents) (Supplementary Fig. 1C, middle and upper panels respectively). Moreover, the formation of the LC3B/Src complex was reduced after treatment with all 6 kinase inhibitors, providing biochemical evidence of inhibition of Src's targeting to intracellular puncta that supported the imaging data (Fig. 2C and lysates shown in Supplementary Fig. 1C and 1E). The screen was validated using inhibitors that are known to block general autophagy (3MA [17]) or Src phosphorylation (dasatinib [5]), and these, as expected, blocked visualisation of active Src at autophagic puncta in FAK −/− cells (Supplementary Fig. 1B). For the 6 inhibitors we identified, termed A–F, we wished to take an unbiased view on their mode of action since we believed that they are relatively non-specific kinase inhibitors (details of these inhibitors can be found in Table 1). Next, we examined how these affected the Akt/p70S6K/S6 pathway (and PDK1 upstream) that we knew to control Src's autophagic targeting (Fig. 1).


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

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

Kinase inhibitors of Src-selective autophagy. (A) FAK −/− cells were treated with the inhibitors A–F for 24 h. Cells were fixed and stained with anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Scale bars: 20 μm. (B) Percentage of cells with active Src localising to intracellular structures was quantified and data presented as mean ± s.d. Significance for all treatments is p < 0.01 (n = 3). (C) LC3B was immunoprecipitated from FAK −/− cell lysates treated with kinase inhibitors. Then immunoblotting was performed using anti-p-Src Y416 and anti-LC3B antibodies.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0010: Kinase inhibitors of Src-selective autophagy. (A) FAK −/− cells were treated with the inhibitors A–F for 24 h. Cells were fixed and stained with anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Scale bars: 20 μm. (B) Percentage of cells with active Src localising to intracellular structures was quantified and data presented as mean ± s.d. Significance for all treatments is p < 0.01 (n = 3). (C) LC3B was immunoprecipitated from FAK −/− cell lysates treated with kinase inhibitors. Then immunoblotting was performed using anti-p-Src Y416 and anti-LC3B antibodies.
Mentions: Next we performed a small drug screen using a commercially available kinase inhibitor library. Adherent FAK −/− cells were treated with 80 different kinase inhibitors (Tocriscreen Kinase Inhibitor Toolbox) for 24 h, then fixed and stained with p-Src Y416 antibody, Deep Red Cell Mask and DAPI, so as to visualise autophagosomes and adhesions, cell cytoplasm and nucleus, respectively. Cells were imaged and analysed using an Olympus Scan-R microscope to calculate the number of Src-positive puncta per cell in untreated FAK-WT (less than 15 puncta per cell) and untreated FAK −/− (58 puncta per cell) cells as negative and positive controls. We focused on 6 chemical entities that significantly reduced the number of Src-positive puncta detected per cell (Fig. 2A and quantified in 2B), but were not in the class of chemicals that would inhibit Src phosphorylation per se (for example, there was no significant decrease in either the amount of total or active Src after treatment of FAK-WT cells with these agents) (Supplementary Fig. 1C, middle and upper panels respectively). Moreover, the formation of the LC3B/Src complex was reduced after treatment with all 6 kinase inhibitors, providing biochemical evidence of inhibition of Src's targeting to intracellular puncta that supported the imaging data (Fig. 2C and lysates shown in Supplementary Fig. 1C and 1E). The screen was validated using inhibitors that are known to block general autophagy (3MA [17]) or Src phosphorylation (dasatinib [5]), and these, as expected, blocked visualisation of active Src at autophagic puncta in FAK −/− cells (Supplementary Fig. 1B). For the 6 inhibitors we identified, termed A–F, we wished to take an unbiased view on their mode of action since we believed that they are relatively non-specific kinase inhibitors (details of these inhibitors can be found in Table 1). Next, we examined how these affected the Akt/p70S6K/S6 pathway (and PDK1 upstream) that we knew to control Src's autophagic targeting (Fig. 1).

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