TY - JOUR
T1 - Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1
AU - Czikora, Istvan
AU - Kim, Kyung Mi
AU - Kása, Anita
AU - Bécsi, Bálint
AU - Verin, Alexander D.
AU - Gergely, Pál
AU - Erdodi, Ferenc
AU - Csortos, Csilla
N1 - Funding Information:
This work was supported by grants CNK80709(GP) and K68416 (EF) from the Hungarian Science Research Fund ; RO1-HL080675 (ADV) and RO1-HL67307 (ADV) from NIH; and TÁMOP4.2.2.-08/1-2008-0019 DERMINOVA, TÁMOP4.2.1./B-09/1/KONV-2010-0007 .
PY - 2011/7
Y1 - 2011/7
N2 - 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.
AB - 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.
KW - Moesin
KW - Protein phosphatase 1
KW - Surface plasmon resonance
KW - TIMAP
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UR - http://www.scopus.com/inward/citedby.url?scp=79958075702&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2011.03.011
DO - 10.1016/j.biochi.2011.03.011
M3 - Article
C2 - 21466834
AN - SCOPUS:79958075702
SN - 0300-9084
VL - 93
SP - 1139
EP - 1145
JO - Biochimie
JF - Biochimie
IS - 7
ER -