Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1

István Czikora, Kyung Mi Kim, Anita Kása, Bálint Bécsi, Alexander D. Verin, Pál Gergely, Ferenc Erdodi, Csilla Csortos

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (Ka = 1.80 × 10 6 M-1) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (∼60% inhibition), mono- (∼50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.

Original languageEnglish
Pages (from-to)1139-1145
Number of pages7
JournalBiochimie
Volume93
Issue number7
DOIs
Publication statusPublished - Jul 1 2011

Keywords

  • Moesin
  • Protein phosphatase 1
  • Surface plasmon resonance
  • TIMAP

ASJC Scopus subject areas

  • Biochemistry

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