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Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

Moore SF, Hunter RW, Hers I - J. Thromb. Haemost. (2014)

Bottom Line: PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation.Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt.Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited.

View Article: PubMed Central - PubMed

Affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol, UK.

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IGF-1–stimulated TSC2 phosphorylation is insufficient to activate mTORC1. Washed platelets were stimulated with IGF-1 (100 nmol L−1) for the indicated times (A) or incubated with vehicle (0.2% DMSO), wortmannin (100 nmol L−1), MK-2206 (1 μmol L−1), BIM I (10 μmol L−1), or rapamycin (200 nmol L−1) for 15 min before stimulation with IGF-1 (100 nmol L−1) for 15 min (B). Phosphorylation of the indicated proteins was assessed by Western blotting of either whole cell lysates or immunoprecipitates (IP). Membranes were reprobed for α-tubulin to confirm equal loading.
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fig03: IGF-1–stimulated TSC2 phosphorylation is insufficient to activate mTORC1. Washed platelets were stimulated with IGF-1 (100 nmol L−1) for the indicated times (A) or incubated with vehicle (0.2% DMSO), wortmannin (100 nmol L−1), MK-2206 (1 μmol L−1), BIM I (10 μmol L−1), or rapamycin (200 nmol L−1) for 15 min before stimulation with IGF-1 (100 nmol L−1) for 15 min (B). Phosphorylation of the indicated proteins was assessed by Western blotting of either whole cell lysates or immunoprecipitates (IP). Membranes were reprobed for α-tubulin to confirm equal loading.

Mentions: The mitogen IGF-1 is a classic agonist of the PI3 kinase/Akt pathway leading to activation of mTORC1 in many cell types 19,20. In platelets, IGF-1 stimulated transient phosphorylation of TSC2 on Ser939 and Thr1462, which closely followed phosphorylation of Akt Ser473 and the Akt substrate PRAS40 (Fig. 3A). Interestingly, despite robust TSC2 phosphorylation, we were unable to detect activation of mTORC1 by observation of either pThr389 p70S6K (Fig. 3A) or pSer65 4E-BP1 (data not shown), suggesting that TSC2 phosphorylation is not sufficient to activate mTORC1. In contrast to thrombin, IGF-1–stimulated TSC2 phosphorylation was completely dependent on PI3 kinase/Akt and ablated by both wortmannin and MK-2206 (Fig. 3B). These results demonstrate that IGF-1 stimulates Akt-dependent TSC2 phosphorylation but that this is insufficient to activate mTORC1 in human platelets.


Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

Moore SF, Hunter RW, Hers I - J. Thromb. Haemost. (2014)

IGF-1–stimulated TSC2 phosphorylation is insufficient to activate mTORC1. Washed platelets were stimulated with IGF-1 (100 nmol L−1) for the indicated times (A) or incubated with vehicle (0.2% DMSO), wortmannin (100 nmol L−1), MK-2206 (1 μmol L−1), BIM I (10 μmol L−1), or rapamycin (200 nmol L−1) for 15 min before stimulation with IGF-1 (100 nmol L−1) for 15 min (B). Phosphorylation of the indicated proteins was assessed by Western blotting of either whole cell lysates or immunoprecipitates (IP). Membranes were reprobed for α-tubulin to confirm equal loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4238809&req=5

fig03: IGF-1–stimulated TSC2 phosphorylation is insufficient to activate mTORC1. Washed platelets were stimulated with IGF-1 (100 nmol L−1) for the indicated times (A) or incubated with vehicle (0.2% DMSO), wortmannin (100 nmol L−1), MK-2206 (1 μmol L−1), BIM I (10 μmol L−1), or rapamycin (200 nmol L−1) for 15 min before stimulation with IGF-1 (100 nmol L−1) for 15 min (B). Phosphorylation of the indicated proteins was assessed by Western blotting of either whole cell lysates or immunoprecipitates (IP). Membranes were reprobed for α-tubulin to confirm equal loading.
Mentions: The mitogen IGF-1 is a classic agonist of the PI3 kinase/Akt pathway leading to activation of mTORC1 in many cell types 19,20. In platelets, IGF-1 stimulated transient phosphorylation of TSC2 on Ser939 and Thr1462, which closely followed phosphorylation of Akt Ser473 and the Akt substrate PRAS40 (Fig. 3A). Interestingly, despite robust TSC2 phosphorylation, we were unable to detect activation of mTORC1 by observation of either pThr389 p70S6K (Fig. 3A) or pSer65 4E-BP1 (data not shown), suggesting that TSC2 phosphorylation is not sufficient to activate mTORC1. In contrast to thrombin, IGF-1–stimulated TSC2 phosphorylation was completely dependent on PI3 kinase/Akt and ablated by both wortmannin and MK-2206 (Fig. 3B). These results demonstrate that IGF-1 stimulates Akt-dependent TSC2 phosphorylation but that this is insufficient to activate mTORC1 in human platelets.

Bottom Line: PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation.Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt.Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited.

View Article: PubMed Central - PubMed

Affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol, UK.

Show MeSH
Related in: MedlinePlus