Limits...
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

Src trafficking to autophagosomes is dependent upon p70S6K. (A) Cell lysates from SCC FAK-WT and FAK −/− cells were immunoblotted with anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6 and anti-GAPDH. (B) Graph shows the relative ratio of p-p70S6K/total p70S6K, p-S6 S235/236/total S6 and p-Akt S473/total Akt in FAK-WT and FAK −/− cells. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 8). (C) FAK −/− cells were treated with the p70S6K inhibitor PF4708671 (10 μM) for 24 h. Cell lysates were immunoblotted with anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-Src Y416, anti-Src and anti-GAPDH. (D) Cells were also fixed and stained for anti-p-Src Y416 (green) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3). (E) LC3B was immunoprecipitated from FAK −/− cells treated with PF4708671 and immunoblotting performed with anti-p-Src Y416 and anti-LC3B. (F) FAK −/− cells were transfected with either 80 nM Scrambled, p-70 S6K 1 or p-70 S6K 2 siRNA for 48 h. Immunoblotting carried out using anti-p70S6K and anti-GAPDH antibodies. (G) Cells were also fixed and stained for anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Scale bars: 20 μm. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4508348&req=5

f0005: Src trafficking to autophagosomes is dependent upon p70S6K. (A) Cell lysates from SCC FAK-WT and FAK −/− cells were immunoblotted with anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6 and anti-GAPDH. (B) Graph shows the relative ratio of p-p70S6K/total p70S6K, p-S6 S235/236/total S6 and p-Akt S473/total Akt in FAK-WT and FAK −/− cells. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 8). (C) FAK −/− cells were treated with the p70S6K inhibitor PF4708671 (10 μM) for 24 h. Cell lysates were immunoblotted with anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-Src Y416, anti-Src and anti-GAPDH. (D) Cells were also fixed and stained for anti-p-Src Y416 (green) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3). (E) LC3B was immunoprecipitated from FAK −/− cells treated with PF4708671 and immunoblotting performed with anti-p-Src Y416 and anti-LC3B. (F) FAK −/− cells were transfected with either 80 nM Scrambled, p-70 S6K 1 or p-70 S6K 2 siRNA for 48 h. Immunoblotting carried out using anti-p70S6K and anti-GAPDH antibodies. (G) Cells were also fixed and stained for anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Scale bars: 20 μm. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3).

Mentions: In examining effects of FAK depletion upon signalling pathways, we found that phosphorylation of Akt (S473), p70S6K (T389) and its downstream target S6 (S235/236 and S240/244) were all reduced in FAK −/− SCCs, while phosphorylation of mTOR (S2448; a key determinant of mTOR activation [12] or its auto-phosphorylation site (2481)) were unaffected (Fig. 1A; reductions quantified in Fig. 1B). This shows that signalling to Akt, p70S6K and S6 was at least partly dependent on FAK in SCC cells.


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

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

Src trafficking to autophagosomes is dependent upon p70S6K. (A) Cell lysates from SCC FAK-WT and FAK −/− cells were immunoblotted with anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6 and anti-GAPDH. (B) Graph shows the relative ratio of p-p70S6K/total p70S6K, p-S6 S235/236/total S6 and p-Akt S473/total Akt in FAK-WT and FAK −/− cells. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 8). (C) FAK −/− cells were treated with the p70S6K inhibitor PF4708671 (10 μM) for 24 h. Cell lysates were immunoblotted with anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-Src Y416, anti-Src and anti-GAPDH. (D) Cells were also fixed and stained for anti-p-Src Y416 (green) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3). (E) LC3B was immunoprecipitated from FAK −/− cells treated with PF4708671 and immunoblotting performed with anti-p-Src Y416 and anti-LC3B. (F) FAK −/− cells were transfected with either 80 nM Scrambled, p-70 S6K 1 or p-70 S6K 2 siRNA for 48 h. Immunoblotting carried out using anti-p70S6K and anti-GAPDH antibodies. (G) Cells were also fixed and stained for anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Scale bars: 20 μm. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0005: Src trafficking to autophagosomes is dependent upon p70S6K. (A) Cell lysates from SCC FAK-WT and FAK −/− cells were immunoblotted with anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6 and anti-GAPDH. (B) Graph shows the relative ratio of p-p70S6K/total p70S6K, p-S6 S235/236/total S6 and p-Akt S473/total Akt in FAK-WT and FAK −/− cells. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 8). (C) FAK −/− cells were treated with the p70S6K inhibitor PF4708671 (10 μM) for 24 h. Cell lysates were immunoblotted with anti-p-Akt S473, anti-Akt, anti-p-p70S6K T389, anti-p70S6K, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-Src Y416, anti-Src and anti-GAPDH. (D) Cells were also fixed and stained for anti-p-Src Y416 (green) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3). (E) LC3B was immunoprecipitated from FAK −/− cells treated with PF4708671 and immunoblotting performed with anti-p-Src Y416 and anti-LC3B. (F) FAK −/− cells were transfected with either 80 nM Scrambled, p-70 S6K 1 or p-70 S6K 2 siRNA for 48 h. Immunoblotting carried out using anti-p70S6K and anti-GAPDH antibodies. (G) Cells were also fixed and stained for anti-p-Src Y416 (green), anti-paxillin (red) and DAPI (blue). Broken arrows show active Src localising to puncta while solid arrows indicate active Src localising to adhesions. Scale bars: 20 μm. Quantification shows the percentage of cells with active Src localising to intracellular puncta. Results are presented as mean ± s.d. and significance is p < 0.01 (n = 3).
Mentions: In examining effects of FAK depletion upon signalling pathways, we found that phosphorylation of Akt (S473), p70S6K (T389) and its downstream target S6 (S235/236 and S240/244) were all reduced in FAK −/− SCCs, while phosphorylation of mTOR (S2448; a key determinant of mTOR activation [12] or its auto-phosphorylation site (2481)) were unaffected (Fig. 1A; reductions quantified in Fig. 1B). This shows that signalling to Akt, p70S6K and S6 was at least partly dependent on FAK in SCC cells.

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