Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.
TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements.
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, Debrecen H-4032, Hungary.
Anita
A
Boratkó
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, Debrecen H-4032, Hungary.
Margit
M
Péter
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, Debrecen H-4032, Hungary.
Zsófia
Z
Thalwieser
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, Debrecen H-4032, Hungary.
Előd
E
Kovács
Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, Debrecen H-4032, Hungary. Electronic address: csortos@med.unideb.hu.
Csilla
C
Csortos
D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002478:Cells, Cultured; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D020648:Peptide Elongation Factor 1; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D054645:Protein Phosphatase 1 / Q000502:physiology*; D011499:Protein Processing, Post-Translational
C094814:EEF1A1 protein, human; D008565:Membrane Proteins; C459597:PPP1R16B protein, human; D020648:Peptide Elongation Factor 1; D054645:Protein Phosphatase 1
Endothelial cell; Protein phosphatase 1; TIMAP; eEF1A1