PP2A Affects Angiogenesis via Its Interaction with a Novel Phosphorylation Site of TSP1.

Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α-TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α.

Dephosphorylation of annexin A2 by protein phosphatase 1 regulates endothelial cell barrier.

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.

Ser69 phosphorylation of TIMAP affects endothelial cell migration.

PURPOSE/AIM: TIMAP (TGF-ß-inhibited membrane-associated protein) is a regulatory subunit of protein phosphatase 1 (PP1). The N-terminal region contains a binding motif for the catalytic subunit of PP1 (PP1c) and a nuclear localization signal (NLS). Phosphorylation of TIMAP on Ser331, Ser333 and Ser337 side chains was shown to regulate the activity of the TIMAP-PP1c complex. Several studies, however, reported an additional side chain of TIMAP. Ser69 is located near to the PP1c binding motif and NLS, therefore, we hypothesized that the phosphorylation of this side chain perhaps may regulate the interaction between TIMAP and PP1c, or may affect the nuclear transport of TIMAP. Materials and Methods: To study the significance of Ser69 phosphorylation, GST-tagged or c-myc-tagged wild type, phosphomimic S69D and phosphonull S69A recombinant TIMAP proteins were expressed in bacteria or endothelial cells, respectively. Protein-protein interactions of the wild type or mutant forms of TIMAP were studied by pull-down and Western blot. Localization of TIMAP S69 mutants in pulmonary artery endothelial cells was detected by immunofluorescent staining and expression and localization of the recombinants were investigated by subcellular fractionation and Western blot. Results: Modifications of Ser69 of TIMAP had no effect on binding of PP1c, ERM or RACK1. However, S69D TIMAP showed enhanced membrane localization and an increased number of membrane protrusions were observed in the cells overexpressing this phosphomimic mutant. Furthermore, significantly faster wound healing and migration rate of the S69D mutant overexpressing cells were detected by endothelial barrier resistance measurements (ECIS). Specific interaction was shown between TIMAP and polo-like kinase 4 (PLK4), a potential kinase to phosphorylate Ser69. Conclusions: Altogether, our results indicate that Ser69 phosphorylation by PLK4 may evoke an enrichment of TIMAP in the plasma membrane region and may play an important role in endothelial cell migration without affecting the PP1c binding ability of TIMAP.

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

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-B55α 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-B55α. 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. Ser315 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 Ser315 in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser315 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.

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington's disease (HD) research.