Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells

2021 ◽  
Author(s):  
Shiyou Liu ◽  
Tsubasa S. Matsui ◽  
Na Kang ◽  
Shinji Deguchi
Keyword(s):  
Replicative Senescence ◽  
Human Fibroblasts ◽  
Elongation Factor ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Elongation Factor 2 ◽  
Protein Components

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

scholarly journals Proteome of actin stress fibers

2021 ◽  
Author(s):  
Shiyou Liu ◽  
Tsubasa S. Matsui ◽  
Na Kang ◽  
Shinji Deguchi
Keyword(s):  
Molecular Basis ◽  
Replicative Senescence ◽  
Human Fibroblasts ◽  
Elongation Factor ◽  
Stress Fibers ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Elongation Factor 2 ◽  
Protein Components

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 263 proteins are associated with SFs, and 101 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

scholarly journals Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

2018 ◽  
Vol 293 (14) ◽  
pp. 5220-5229 ◽  
Author(s):  
Denis Susorov ◽  
Nikita Zakharov ◽  
Ekaterina Shuvalova ◽  
Alexander Ivanov ◽  
Tatiana Egorova ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Elongation Factor 2

scholarly journals Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2

2019 ◽  
Vol 400 (4) ◽  
pp. 501-512 ◽  
Author(s):  
Rita Mateus-Seidl ◽  
Sebastian Stahl ◽  
Stefan Dengl ◽  
Fabian Birzele ◽  
Hedda Herrmuth ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Side Chain ◽  
Translation Elongation Factor ◽  
Inhibit Protein Synthesis ◽  
Transcriptional Responses ◽  
Translation Elongation ◽  
Structural Alterations ◽  
Eukaryotic Translation ◽  
Adp Ribosylation ◽  
Elongation Factor 2

Abstract The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 ‘modifier region’ triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.

scholarly journals Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes

1998 ◽  
Vol 336 (3) ◽  
pp. 525-529 ◽  
Author(s):  
Tricia A. DIGGLE ◽  
Nicholas T. REDPATH ◽  
Kate J. HEESOM ◽  
Richard M. DENTON
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Polypeptide Chain ◽  
Elongation Factor ◽  
Chain Elongation ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Elongation Factor 2 ◽  
Fold Activation

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 µM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8–10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 µM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.

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