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Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

ABSTRACT: The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ?50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

SUBMITTER: Kearney AL

PROVIDER: S-EPMC6851323 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

Kearney Alison L AL Cooke Kristen C KC Norris Dougall M DM Zadoorian Armella A Krycer James R JR Fazakerley Daniel J DJ Burchfield James G JG James David E DE

The Journal of biological chemistry 20190922 45

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we dire ...[more]

PMID: 31548312

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Project description:ObjectiveThe inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity.Methods and resultsWe provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine.ConclusionTaken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.

Project description:BackgroundCampylobacter jejuni causes acute disease characterized by severe diarrhea containing blood and leukocytes, fever, and abdominal cramping. Disease caused by C. jejuni is dependent on numerous bacterial and host factors. C. jejuni invasion of the intestinal epithelial cells is seen in both clinical samples and animal models indicating that host cell invasion is, in part, necessary for disease. C. jejuni utilizes a flagellar Type III Secretion System (T3SS) to deliver the Campylobacter invasion antigens (Cia) to host cells. The Cia proteins modulate host cell signaling leading to actin cytoskeleton rearrangement necessary for C. jejuni host cell invasion, and are required for the development of disease.ResultsThis study was based on the hypothesis that the C. jejuni CiaD effector protein mediates Erk 1/2 dependent cytoskeleton rearrangement. We showed that CiaD was required for the maximal phosphorylation of Erk 1/2 by performing an immunoblot with a p-Erk 1/2 specific antibody and that Erk 1/2 participates in C. jejuni invasion of host cells by performing the gentamicin protection assay in the presence and absence of the PD98059 (a potent inhibitor of Erk 1/2 activation). CiaD was also found to be required for the maximal phosphorylation of cortactin S405 and S418, as judged by immunoblot analysis. The response of human INT 407 epithelial cells to infection with C. jejuni was evaluated by confocal microscopy and scanning electron microscopy to determine the extent of membrane ruffling. This analysis revealed that CiaD, Erk 1/2, and cortactin participate in C. jejuni-induced membrane ruffling. Finally, cortactin and N-WASP were found to be involved in C. jejuni invasion of host cells using siRNA to N-WASP, and siRNA to cortactin, coupled with the gentamicin protection assay.ConclusionWe conclude that CiaD is involved in the activation of Erk 1/2 and that activated Erk 1/2 facilitates C. jejuni invasion by phosphorylation of cortactin on serine 405 and 418. This is the first time that cortactin and N-WASP have been shown to be involved in C. jejuni invasion of host cells. These data also provide a mechanistic basis for the requirement of Erk 1/2 in C. jejuni-mediated cytoskeletal rearrangement.

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Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

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Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

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