Thr2446Is a Novel Mammalian Target of Rapamycin (mTOR) Phosphorylation Site Regulated by Nutrient Status

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70S6K activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70S6K activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70S6K activity induced by insulin and leucine correlated with changes in phosphorylation of Thr389, the mTOR phosphorylation site on p70S6K, and of Ser2448 on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70S6K, leading to the absence of p70S6K activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70S6K, suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70S6K. We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70S6K pathway requires PDK1 in a way that differs from that of insulin.

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The kinase triad, AMPK, mTORC1 and ULK1, maintains energy and nutrient homoeostasis

Biochemical Society Transactions ◽

10.1042/bst20130030 ◽

2013 ◽

Vol 41 (4) ◽

pp. 939-943 ◽

Cited By ~ 65

Author(s):

Elaine A. Dunlop ◽

Andrew R. Tee

Keyword(s):

Amino Acid ◽

Cell Growth ◽

Protein Kinases ◽

Mammalian Target Of Rapamycin ◽

Nutrient Status ◽

Protein Translation ◽

Cellular Growth ◽

Target Of Rapamycin ◽

Amp Activated Protein Kinase ◽

Rate Limiting

In order for cells to divide in a proficient manner, they must first double their biomass, which is considered to be the main rate-limiting phase of cell proliferation. Cell growth requires an abundance of energy and biosynthetic precursors such as lipids and amino acids. Consequently, the energy and nutrient status of the cell is acutely monitored and carefully maintained. mTORC1 [mammalian (or mechanistic) target of rapamycin complex 1] is often considered to be the master regulator of cell growth that enhances cellular biomass through up-regulation of protein translation. In order for cells to control cellular homoeostasis during growth, there is close signalling interplay between mTORC1 and two other protein kinases, AMPK (AMP-activated protein kinase) and ULK1 (Unc-51-like kinase 1). This kinase triad collectively senses the energy and nutrient status of the cell and appropriately dictates whether the cell will actively favour energy- and amino-acid-consuming anabolic processes such as cellular growth, or energy- and amino-acid-generating catabolic processes such as autophagy. The present review discusses important feedback mechanisms between these three homoeostatic protein kinases that orchestrate cell growth and autophagy, with a particular focus on the mTORC1 component raptor (regulatory associated protein of mammalian target of rapamycin), as well as the autophagy-initiating kinase ULK1.

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The serine/threonine kinase ULK1 is a target of multiple phosphorylation events

Biochemical Journal ◽

10.1042/bj20101894 ◽

2011 ◽

Vol 440 (2) ◽

pp. 283-291 ◽

Cited By ~ 133

Author(s):

Markus Bach ◽

Mark Larance ◽

David E. James ◽

Georg Ramm

Keyword(s):

Mammalian Target Of Rapamycin ◽

Phosphorylation Site ◽

Degradation Process ◽

Dominant Negative ◽

Serine Threonine Kinase ◽

Kinase Activation ◽

Threonine Kinase ◽

Convergence Point ◽

Mtor Phosphorylation

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser555in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser774 and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr180 that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.

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