Post‐translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface

Abstract

Annexin A1 (ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells. LPS caused serine-phosphorylation of ANXA1 (ANXA1-S 27 -PO 4 ) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild-type or mutated (S 27 /A) ANXA1 constructs tagged with enhanced green fluorescence protein.The trafficking of ANXA1-S 27 -PO 4 to the cell surface was dependent on PI3-kinase and MAPkinase. It also required HMG-coenzyme A and myristoylation. The effects of HMG-coenzyme A blockade were overcome by mevalonic acid (the product of HMG-coenzyme A) and farnesylpyrophosphate but not by geranyl-geranylpyrophosphate or cholesterol. Together, these results suggest that serine-27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters). Keywordsphosphorylation; green fluorescence protein; lipidation; signaling Annexin A1 (ANXA1) is a Ca 2+ -and phospholipids-binding protein that acts both intracellularly and extracellularly to modulate cell signaling. Data from our laboratory have shown that it has a particularly important role in the regulation of the host defense and neuroendocrine systems and that ANXA1 mimetics may provide a novel therapeutic approach to the control of inflammatory disease (reviewed in ref 1). The regulatory actions of ANXA1 in both systems appear to be exerted mainly extracellularly, via membrane-bound cell surface receptors (2-4). Access of endogenous ANXA1 to these receptors is thus dependent on the translocation of the protein from its cytoplasmic stores to the cell surface (1). These conclusions are supported by evidence that within the neuroendocrine system ANXA1 is expressed in cells adjacent to its target cells (5), and it is concentrated at sites where the cells make contact with UKPMC Funders Group Author ManuscriptUKPMC Funders Group Author Manuscript endocrine cells (6) and translocated to the cell surface in response to appropriate stimuli (7,8). Further evidence of the paracrine/juxtacrine mode of ANXA1 action is provided by evidence that drugs that prevent the cellular exportation of the protein ablate the regulatory actions of endogenous ANXA1 within the neuroendocrine system (6,7,9,10) as also do compounds that destroy the cell surface ANXA1 receptors (2). Substantial evidence...

Identification of an essential endogenous regulator of blood–brain barrier integrity, and its pathological and therapeutic implications

“…Stable hCMEC/D3 cell lines were produced by transfection with an empty pRc/CMV plasmid or constructs bearing the full length or an antisense sequence for human ANXA1 (10) using HiFect according to manufacturer instructions (Lonza). Cells were screened for analysis of ANXA1 expression by flow cytometry and for expression of the neomycin resistance cassette by RT-PCR according to standard protocols (44).…”

Annexin A1 regulates EGFR activity and alters EGFR-containing tumour-derived exosomes in head and neck cancers

“…In contrast to the mainly antiinflammatory functions of secreted ANXA1, the fulllength intracellular protein has been associated with a wide range of cellular functions such as cytoskeletal organisation, plasma membrane repair, autophagy and intracellular calcium signalling as well as lipid-mediated signalling pathways [8e11]. Different intracellular localisations and stimulus-or modification-dependent redistributions of the ANXA1 protein have been observed [12,13]. A role of ANXA1 in tumour development and progression has been described for different cancer subtypes.…”

Anti-Inflammatory Mechanisms of the Annexin A1 Protein and Its Mimetic Peptide Ac2-26 in Models of Ocular Inflammation In Vivo and In Vitro

“…The N-terminal domain is unique for each member of the superfamily, and several investigations characterized this region as a promoter of anti-inflammatory actions (16,19,20).…”

“…Moreover, endogenous AnxA1's access to membrane receptors appears to depend on phosphorylation processes that allow translocation of the protein from the cytoplasm to the cell surface (20,22).…”