Identification of a Proline-rich Akt Substrate as a 14-3-3 Binding Partner

Kovacina, Kristina S.; Park, Grace Y.; Bae, Sun Sik; Guzzetta, Andrew W.; Schaefer, Erik; Birnbaum, Morris J.; Roth, Richard A.

doi:10.1074/jbc.m210837200

Cited by 329 publications

(355 citation statements)

References 25 publications

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“…Myc-tagged plasmid of PRAS40 was a kind gift from Dr Richard A Roth and was generated by subcloning the entire coding region of human cDNA (GenBank accession number BC007416, IMAGE clone number 2988648) into a pcDNA3.1( + ) vector (Invitrogen, Carlsbad, CA, USA) (Kovacina et al, 2003). Both the PRAS40 plasmid and plasmid pcDNA3.1( + ) vector were grown overnight, and plasmid was extracted using a Qiagen kit (Valencia, CA, USA).…”

Section: Liposome-mediated Pras40 Complementary Dna Transfectionmentioning

confidence: 99%

Increased Expression of a Proline-Rich Akt Substrate (PRAS40) in Human Copper/Zinc-Superoxide Dismutase Transgenic Rats Protects Motor Neurons from Death after Spinal Cord Injury

Narasimhan

Saito

et al. 2007

J Cereb Blood Flow Metab

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The serine-threonine kinase, Akt, plays an important role in the cell survival signaling pathway. A proline-rich Akt substrate, PRAS40, has been characterized, and an increase in phospho-PRAS40 (pPRAS40) is neuroprotective after transient focal cerebral ischemia. However, the involvement of PRAS40 in the cell death/survival pathway after spinal cord injury (SCI) is unclear. Liposomemediated PRAS40 transfection was performed to study whether overexpression of pPRAS40 is neuroprotective. We further examined the expression of pPRAS40 after SCI by immunohistochemistry and Western blot using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats and wild-type (Wt) littermates. We then examined the relationship between PRAS40 and Akt by injection of LY294002, a phosphatidylinositol 3-kinase (PI3K) pathway inhibitor, or Akt inhibitor IV, a compound that inhibits Akt activation after SCI. Our data demonstrated that increased pPRAS40 resulted in survival of more motor neurons compared with control complementary DNA transfection. Phosphorylated PRAS40 increased in the Wt rats after SCI, whereas there was a greater and prolonged increase in the SOD1 Tg rats. Coimmunoprecipitation showed that binding of pPRAS40 with 14-3-3 increased 1 day after SCI in the Wt rats, whereas there was a significant increase in the Tg rats. The inhibitor studies showed that phospho-Akt and pPRAS40 were decreased after injection of LY294002 or Akt inhibitor IV. We conclude that an increase in pPRAS40 by transfection after SCI results in survival of motor neurons, and overexpression of SOD1 in the Tg rats results in an increase in endogenous pPRAS40 and a decrease in motor neuron death through the PI3K/Akt pathway.

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Section: Liposome-mediated Pras40 Complementary Dna Transfectionmentioning

confidence: 99%

Increased Expression of a Proline-Rich Akt Substrate (PRAS40) in Human Copper/Zinc-Superoxide Dismutase Transgenic Rats Protects Motor Neurons from Death after Spinal Cord Injury

Narasimhan

Saito

et al. 2007

J Cereb Blood Flow Metab

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“…Materials-Antibodies to raptor (20), 4E-BP1 (21), Akt1 (22), S6K1 (23), PRAS40 (14), HA (20), and phosphospecific antibodies to the Thr-36 and Thr-45 sites (24) have been described previously. Phosphospecific antibodies to the Thr-389 site in S6K1 were from Cell Signaling Technology, Inc.…”

Section: Methodsmentioning

confidence: 99%

“…The inhibitory activity of PRAS40 is suggested to occur by direct competition with 4E-BP1 and S6K1 for binding to raptor (11,12). PRAS40 was originally isolated with a 14-3-3 affinity column, and its binding with 14-3-3 depends on Akt/protein kinase B-mediated phosphorylation at Thr-246 (14). PRAS40 binding to 14-3-3 also depends on nutrient sufficiency and is inhibited by treatment with rapamycin, the specific inhibitor against mTORC1 (13,15).…”

mentioning

confidence: 99%

Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

Wang

Harris

Lawrence³

2008

Journal of Biological Chemistry

164

143

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The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [ 32 P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/ protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition. Mammalian target of rapamycin (mTOR)2 has been demonstrated as a key element in signaling pathways controlling cell size, proliferation, and metabolism (1, 2). Two mTOR signaling complexes, mTORC1 and mTORC2, have been discovered. The rapamycin-sensitive mTORC1 consists of the catalytic subunit mTOR, the substrate-binding subunit raptor (regulatory associated protein of mTOR), mLST8 (also known as G␤L), and PRAS40 (1, 2) and controls protein translation (1, 2). mTORC2, the rapamycin-insensitive form, contains mTOR, rictor, SIN1, mLST8, and PRR5 (1, 2) and functions as a PDK2 (phosphoinositide-dependent protein kinase 2) to phosphorylate Akt/protein kinase B at Ser-473 and regulates the actin cytoskeleton (1, 2). The best characterized downstream effectors of mTORC1 are S6K1 and 4E-BP1 (also known as PHAS-I), both of which are phosphorylated by mTORC1 at multiple sites and are involved in the control of mRNA translation (1, 2). The nature of the phosphorylation sites in these two mTORC1 substrates is surprisingly different: either (S/T)P (3) or h(S/T)h (where h represents hydrophobic) (4). Thus, the surrounding amino acids appear not to be the major determinant for phosphorylation. A critical motif called the TOR signaling (TOS) motif has been discovered in the NH 2 terminus of S6K1 (FDIDL) and COOH terminus of 4E-BP1 (FEMDI) (5, 6). Mutation of the TOS motif not only decreases the rate of phosphorylation of S6K1 and 4E-BP1 by mTOR but also disrupts interaction between these substrates and raptor (5-8).PRAS40 has been recently identified as a protein associated with mTORC1 (9, 10). PRAS40 predominantly interacts with raptor, althoug...

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“…Prior to the current study, the only reported function for PRAS40 was its binding to the Raptor (regulatory-associated protein of mTOR) subunit and inhibiting mTORC1 activation (20,23,28,29,49). Therefore, we tested whether downregulation of PRAS40 alone leads to mTORC1 activation and, more importantly, whether Raptor, mTOR, and 14-3-3 have any roles in stress-triggered exosome secretion.…”

mentioning

confidence: 99%

“…The proline-rich Akt substrate of 40 kDa (PRAS40) was initially identified as a direct substrate of Akt kinase and a binding partner for the 14-3-3 scaffolding molecule (20). Most studies focused on PRAS40's role in insulin, as well as nerve growth factor (NGF) and platelet-derived growth factor (PDGF), signaling to the mTOR (mammalian target of rapamycin) pathway (specifically mTORC1), which regulates cell metabolism, protein synthesis, and cell growth (21)(22)(23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

PRAS40 Connects Microenvironmental Stress Signaling to Exosome-Mediated Secretion

Guo

Jayaprakash

Jian

et al. 2017

Molecular and Cellular Biology

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Secreted exosomes carrying lipids, proteins, and nucleic acids conduct cell-cell communications within the microenvironment of both physiological and pathological conditions. Exosome secretion is triggered by extracellular or intracellular stress signals. Little is known, however, about the signal transduction between stress cues and exosome secretion. To identify the linker protein, we took advantage of a unique finding in human keratinocytes. In these cells, although transforming growth factor alpha (TGF-␣) and epidermal growth factor (EGF) share the same EGF receptor and previously indistinguishable intracellular signaling networks, only TGF-␣ stimulation causes exosome-mediated secretion. However, deduction of EGF-activated pathways from TGF␣-activated pathways in the same cells allowed us to identify the proline-rich Akt substrate of 40 kDa (PRAS40) as the unique downstream effector of TGF-␣ but not EGF signaling via threonine 308-phosphorylated Akt. PRAS40 knockdown (KD) or PRAS40 dominant-negative (DN) mutant overexpression blocks not only TGF-␣-but also hypoxia-and H 2 O 2 -induced exosome secretion in a variety of normal and tumor cells. Site-directed mutagenesis and gene rescue studies show that Akt-mediated activation of PRAS40 via threonine 246 phosphorylation is both necessary and sufficient to cause exosome secretion without affecting the endoplasmic reticulum/Golgi pathway. Identification of PRAS40 as a linker protein paves the way for understanding how stress regulates exosome secretion under pathophysiological conditions.

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Identification of a Proline-rich Akt Substrate as a 14-3-3 Binding Partner

Cited by 329 publications

References 25 publications

Increased Expression of a Proline-Rich Akt Substrate (PRAS40) in Human Copper/Zinc-Superoxide Dismutase Transgenic Rats Protects Motor Neurons from Death after Spinal Cord Injury

Increased Expression of a Proline-Rich Akt Substrate (PRAS40) in Human Copper/Zinc-Superoxide Dismutase Transgenic Rats Protects Motor Neurons from Death after Spinal Cord Injury

Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

PRAS40 Connects Microenvironmental Stress Signaling to Exosome-Mediated Secretion

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