Dataset Information

Human Keratinocyte Differentiation Requires Translational Control by the eIF2? Kinase GCN2.

ABSTRACT: Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the ? subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2? kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2? phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2? phosphorylation by the general control nonderepressible 2 (GCN2) protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2? and translational control in the formation of an intact human epidermis.

SUBMITTER: Collier AE

PROVIDER: S-EPMC5873978 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2.

Collier Ann E AE Wek Ronald C RC Spandau Dan F DF

The Journal of investigative dermatology 20170517 9

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2α kinases function in the integrative stress response to inhibit general pro ...[more]

PMID: 28528168

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Project description:BackgroundWhen eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2 Delta counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism.ResultsWhile the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2 Delta strain showed slow growth in media devoid of only Trp or Arg. Consistent with the growth phenotypes, profiles of genome-wide tRNA charging revealed significant decrease in cognate tRNA charging only in the gcn2 Delta strain upon depletion for Trp or Arg. In contrast, there was no change in tRNA charging during His and Leu depletion in either the wild-type or gcn2 Delta strains, consistent with the null effect on growth during loss of these amino acids. We determined that the growth phenotype of Trp depletion is derived from feedback inhibition of aromatic amino acid biosynthesis. By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased. The growth phenotype of Arg depletion is derived from unbalanced nitrogen metabolism. By supplementing ornithine upon Arg depletion, both growth and tRNAArg charging were partially restored.ConclusionUnder mild stress conditions the basal activity of Gcn2p is sufficient to allow for proper adaptation to amino acid depletion. This study highlights the importance of the GCN2 eIF2 kinase pathway for maintaining metabolic homeostasis, contributing to appropriate tRNA charging and growth adaptation in response to culture conditions deficient for the central amino acids, tryptophan and arginine.

Dataset Information

Human Keratinocyte Differentiation Requires Translational Control by the eIF2? Kinase GCN2.

Publications

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2.

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