Protein phosphatase 2A–mediated flotillin-1 dephosphorylation up-regulates endothelial cell migration and angiogenesis regulation

Abstract

Edited by Henrik G. Dohlman Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55␣ holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH S-transferase-tagged PP2A-B55␣. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser 315 is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser 315 in flotillin-1 significantly affects its interaction with PP2A-B55␣ and that PP2A-B55␣ dephosphorylates phospho-Ser 315. Spreading, attachment, migration, and in vitro tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…Several other serine residues of flotillin-1 and -2 are predicted by large-scale phosphoproteomic studies to undergo phosphorylation [131,132], but the physiological role of these modifications has not been studied. Flotillin-1 phosphorylated on Ser315 dissociates from the plasma membrane and apparently localizes to the cytosol [38,130]. In fact, in pulmonary artery epithelial cells, the majority of flotillin-1 was cytosolic [130], contradicting the established image of flotillins as (exclusively) membrane-bound proteins.…”

“…Flotillin-1 phosphorylated on Ser315 dissociates from the plasma membrane and apparently localizes to the cytosol [38,130]. In fact, in pulmonary artery epithelial cells, the majority of flotillin-1 was cytosolic [130], contradicting the established image of flotillins as (exclusively) membrane-bound proteins. Of note, the cytosolic flotillin-1 was not palmitoylated [38].…”

“…To complete the picture of the unique properties of flotillin-1, one should mention its phosphorylation on Ser315 catalyzed by PKC and dephosphorylation by protein phosphatase 2A (PP2A) [38,130]. Notably, no homologous serine residue is present in flotillin-2.…”

“…Hypothetically, the shift of flotillin-1 to the cytosol could also be linked with reduction of the sphingosine level in the plasma membrane (see next section), which together with the depalmitoylation would deprive flotillin-1 of its membrane anchors ( Figure 3C). In pulmonary artery endothelial cells, only the membrane-bound flotillin-1 enhances cell migration and formation of endothelial barrier [130], most likely after assembling in hetero-oligomers with flotillin-2 at the plasma membrane ( Figure 3C). One should be reminded here that Ser315 phosphorylation of fotilllin-1 by PKC also affects clustering and/or internalization of DAT [38,109], unlike the endocytosis of other cargo requiring tyrosine phosphorylation of flotillins (see Figure 2).…”

The role of lipid species in membranes and cancer-related changes

“…As shown for caveolin, phosphorylation of flotillin also seems to regulate its function. In the case of flotillin, dephosphorylation upregulates endothelial cell migration and angiogenesis [112].…”