Annexin A2 (ANXA2) is a multifunctional protein expressed in nearly all human tissues and cell types, playing a role in various signaling pathways. It is subjected to phosphorylation, but no specific protein phosphatase has been identified in its posttranslational regulation yet. Using pull-down assay followed by liquid chromatography-mass spectrometry analysis we found that ANXA2 interacts with TIMAP (TGF-beta-inhibited membrane-associated protein) in pulmonary artery endothelial cells. TIMAP is highly expressed in endothelial cells, where it acts as a regulatory and targeting subunit of protein phosphatase 1 (PP1). TIMAP plays an important role in the regulation of the endothelial barrier maintenance through the dephosphorylation of its several substrate proteins. In the present work, phosphorylation of Ser25 side chain in ANXA2 by protein kinase C (PKC) was shown both in vivo and in vitro. Phosphorylation level of ANXA2 at Ser25 increased greatly by inhibition of PP1 and by depletion of its regulatory subunit, TIMAP, implying a role of this PP1 holoenzyme in the dephosphorylation of ANXA2. Immunofluorescence staining and subcellular fractionations revealed a diffuse subcellular localization for the endogenous ANXA2, but phospho-Ser25 ANXA2 was mainly detected in the membrane. ANXA2 depletion lowered the basal endothelial barrier and inhibited cell migration, but had no significant effect on cell proliferation or viability. ANXA2 depleted cells failed to respond to PMA treatment, indicating an intimately involvement of phospho-ANXA2 in PKC signaling. Moreover, phosphorylation of ANXA2 disrupted its interaction with S100A10 suggesting a phosphorylation dependent multiple regulatory role of ANXA2 in endothelial cells. Our results demonstrate the pivotal role of PKC-ANXA2-PP1 pathway in endothelial cell signaling, especially in barrier function and cell migration.
Keywords: PKC; annexin A2; protein dephosphorylation; protein phosphatase 1.
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