The arrestin-domain containing protein AdcA is a response element to stress

Cell Communication and Signaling The arrestin-domain containing protein AdcA is a response element to stress Clmence Habourdin 0 1 2 3 4 Grard Klein 0 1 2 3 4 Tsuyoshi Araki 6 Jeffrey G Williams 6 Laurence Aubry 0 1 2 3 4 5 0 Universite Joseph Fourier , F-38041 Grenoble , France 1 CEA, iRTSV, Laboratoire Biologie a Grande Echelle , F-38054 Grenoble , France 2 INSERM, U1038 , F-38054 Grenoble , France 3 Universite Joseph Fourier , F-38041 Grenoble , France 4 CEA, iRTSV, Laboratoire Biologie a Grande Echelle , F-38054 Grenoble , France 5 CEA-Grenoble, Institut de Recherche en Technologies et Sciences pour le Vivant, Laboratoire de Biologie a Grande Echelle , Equipe OdyCell, 17 rue des Martyrs, 38054 Grenoble Cedex 09 , France 6 College of Life Sciences, Welcome Trust Biocentre, University of Dundee , DD1 5EH, Dundee , United Kingdom Background: Cell behaviour is tightly determined by sensing and integration of extracellular changes through membrane detectors such as receptors and transporters and activation of downstream signalling cascades. Arrestin proteins act as scaffolds at the plasma membrane and along the endocytic pathway, where they regulate the activity and the fate of some of these detectors. Members of the arrestin clan are widely present from unicellular to metazoa, with roles in signal transduction and metabolism. As a soil amoeba, Dictyostelium is frequently confronted with environmental changes likely to compromise survival. Here, we investigated whether the recently described arrestin-related protein AdcA is part of the cell response to stresses. Results: Our data provide evidence that AdcA responds to a variety of stresses including hyperosmolarity by a transient phosphorylation. Analysis in different mutant backgrounds revealed that AdcA phosphorylation involves pathways other than the DokA and cGMP-dependent osmostress pathways, respectively known to regulate PKA and STATc, key actors in the cellular response to conditions of hyperosmolarity. Interestingly, however, both AdcA and STATc are sensitive to changes in the F-actin polymerization status, suggesting a common primary sensor/ trigger and linking the stress-sensitive kinase responsive for AdcA phosphorylation to the actin cytoskeleton. We also show that STATc-dependent transcriptional activity is involved for the timely dephosphorylation of AdcA in cells under stress. Conclusion: Under osmotic stress, AdcA undergoes a phosphorylation-dephosphorylation cycle involving a stress-sensitive kinase and the transcription regulator STATc. This transient post-transcriptional modification may allow a regulation of AdcA function possibly to optimize the cellular stress response. Arrestin; Stress; STAT transcription factor; Hyperosmolarity; Dictyostelium; Phosphorylation - Background Cells are continuously subjected to environmental cues that determine their behaviour in terms of motility, adhesion, growth and differentiation. Their capacity to sense and respond to external stimuli largely relies on membrane proteins including receptors, adhesion molecules, channels and transporters that function as sensors and signal transducers. In the cytoplasm, adaptor proteins from the arrestin clan play key roles in the signal relay, acting downstream of transmembrane spanning proteins at the plasma membrane or/and along the endocytic pathway. A well-characterized system is the coupling of arrestins to ligand-activated G-protein coupled receptors (GPCRs) allowing their temporal desensitization, their trafficking to specific endocytic compartments and the activation of secondary signalling cascades [1-5]. Over the past few years, a family of arrestin-related proteins have been identified in mammals (Arrdcs) as well as in lower organisms such as fungi (ARTs) and amoebae (Adcs) [6-9]. Recent data indicated that ART adaptor proteins regulate the fate of plasma membrane permeases through a conserved ubiquitination-dependent pathway in response to changes in nutritive or pH conditions. ARTs allow the recruitment of the Rsp5 ubiquitin ligase to the vicinity of membrane targets, leading to their ubiquitination and subsequent degradation [8,10-13]. The functions of Arrdcs are still under investigation but recent studies indicate that these proteins could similarly behave as ubiquitin ligase adaptors allowing the regulated ubiquitination of plasma membrane targets and their ensuing downregulation [14,15]. The social amoeba Dictyostelium harbours 6 arrestindomain containing proteins (AdcA to F). Their expression is highly regulated during the developmental cycle but the role of the Adcs at the different stages of the developmental program is completely enigmatic. In contrast to canonical visual and -arrestins, the Dictyostelium Adcs contain additional functional domains besides the arrestin core, including lipid binding- and protein-protein interaction domains [7]. The AdcA protein is extended on both sides of the arrestin core by an N-terminal histidine-rich domain and a C-terminal FYVE domain. This latter domain contributes to the constitutive association of AdcA to the endocytic pathway [16]. The AdcA protein is most highly expressed during the unicellular growth phase of Dictyostelium. At this stage, cells are faced with a broad range of environmental changes, ranging from hypo- or hyperosmotic conditions to nutrient deprivation and their survival in such conditions relies on multiple and complex strategies. Hyperosmotic challenge activates an intricate transcriptionally regulated osmostress response [17]. Our data presented here provide evidence that AdcA undergoes a massive phosphorylation in response to a variety of stresses, including hypertonicity, and that AdcA dephosphorylation is partly dependent on a STATc pathway and correlates with cell recovery, raising the question of a possible role of AdcA in stress response. Results AdcA is multiphosphorylated in response to sorbitol-induced hyperosmotic stress In preliminary experiments, we observed that changes of external medium (12 mM Na,K-Pi buffer vs HL5 axenic medium) affected the electrophoretic pattern of the AdcA protein. We therefore examined the impact of various cellular stresses especially osmotic stress to determine the signal(s) responsible for this modification. In our assays, exponentially growing cells were centrifuged, resuspended in fresh nutritive medium and left to recover in shaking suspension for at least 1 h 30 prior to stress application. This protocol avoided, thereafter, any possible stress due to centrifugation (transient anaerobiosis, mechanical constrains, temperature shift) and nutrient depletion. In these conditions, in KAx-3 cells, AdcA was detected as a major higher mobility form and occasionally a minor slower form (Figure 1A, bands 1 and 2). Addition of 200 mM sorbitol to the medium led to a massive and transient modification of the protein. Within minutes after sorbitol addition, the electrophoretic mobility of AdcA gradually (...truncated)