Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex

2021 ◽

Vol 7 ◽

Author(s):

Taku Kaitsuka ◽

Kazuhito Tomizawa ◽

Masayuki Matsushita

Keyword(s):

Heat Shock ◽

Protein Phosphatase ◽

Elongation Factor ◽

Protein Phosphatase 1 ◽

Cyclin Dependent Kinase ◽

Proteotoxic Stress ◽

Eukaryotic Elongation Factor ◽

Functional Regulation

Several variant proteins are produced from EEF1D, including two representative proteins produced via alternative splicing machinery. One protein is the canonical translation eukaryotic elongation factor eEF1Bδ1, and the other is the heat shock-responsive transcription factor eEF1BδL. eEF1Bδ1 is phosphorylated by cyclin-dependent kinase 1 (CDK1), but the machinery controlling eEF1BδL phosphorylation and dephosphorylation has not been clarified. In this study, we found that both proteins were dephosphorylated under heat shock and proteotoxic stress, and this dephosphorylation was inhibited by okadaic acid. Using proteins with mutations at putative phosphorylated residues, we revealed that eEF1Bδ1 and eEF1BδL are phosphorylated at S133 and S499, respectively, and these residues are both CDK1 phosphorylation sites. The eEF1BδL S499A mutant more strongly activated HSPA6 promoter-driven reporter than the wild-type protein and S499D mutant. Furthermore, protein phosphatase 1 (PP1) was co-immunoprecipitated with eEF1Bδ1 and eEF1BδL, and PP1 dephosphorylated both proteins in vitro. Thus, this study clarified the role of phosphorylation/dephosphorylation in the functional regulation of eEF1BδL during heat shock.

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Protein Phosphatase 1 Regulates Human Cytomegalovirus Protein Translation by Restraining AMPK Signaling

Frontiers in Microbiology ◽

10.3389/fmicb.2021.698603 ◽

2021 ◽

Vol 12 ◽

Author(s):

Carmen Stecher ◽

Sanja Marinkov ◽

Lucia Mayr-Harting ◽

Ana Katic ◽

Marie-Theres Kastner ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Protein Phosphatase ◽

Catalytic Domain ◽

Human Fibroblasts ◽

Elongation Factor ◽

Early Time ◽

Protein Translation ◽

Protein Phosphatase 1 ◽

Ampk Signaling ◽

Protein Protein Interaction

Human cytomegalovirus (HCMV) carries the human protein phosphatase 1 (PP1) and other human proteins important for protein translation in its tegument layer for a rapid supply upon infection. However, the biological relevance behind PP1 incorporation and its role during infection is unclear. Additionally, PP1 is a difficult molecular target due to its promiscuity and similarities between the catalytic domain of multiple phosphatases. In this study, we circumvented these shortcomings by using 1E7-03, a small molecule protein–protein interaction inhibitor, as a molecular tool of noncatalytic PP1 inhibition. 1E7-03 treatment of human fibroblasts severely impaired HCMV replication and viral protein translation. More specifically, PP1 inhibition led to the deregulation of metabolic signaling pathways starting at very early time points post-infection. This effect was at least partly mediated by the prevention of AMP-activated protein kinase dephosphorylation, leading to elongation factor 2 hyperphosphorylation and reduced translation rates. These findings reveal an important mechanism of PP1 for lytic HCMV infection.

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Activity of protein phosphatases against initiation factor-2 and elongation factor-2

Biochemical Journal ◽

10.1042/bj2720175 ◽

1990 ◽

Vol 272 (1) ◽

pp. 175-180 ◽

Cited By ~ 56

Author(s):

N T Redpath ◽

C G Proud

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Protein Phosphatases ◽

Protein Phosphatase 2A ◽

Elongation Factor ◽

Protein Phosphatase 1 ◽

Initiation Factor ◽

Elongation Factor 2 ◽

Initiation Factor 2 ◽

Phosphatase 2A

The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.

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