PP2A Activation by β2-Adrenergic Receptor Agonists

Abstract

Understanding the mechanisms that regulate cell migration is important for devising novel therapies to control metastasis or enhance wound healing. Previously, we demonstrated that ␤ 2 -adrenergic receptor (␤ 2 -AR) activation in keratinocytes inhibited their migration by decreasing the phosphorylation of a critical promigratory signaling component, the extracellular signal-related kinase (ERK). Here we demonstrate that ␤ 2 -ARinduced inhibition of migration is mediated by the activation of the serine/threonine phosphatase PP2A. Pretreating human keratinocytes with the PP2A inhibitor, okadaic acid, prevented the ␤ 2 -AR-induced inhibition of migration, either as isolated cells or as a confluent sheet of cells repairing an in vitro "wound" and also prevented the ␤ 2 -AR-induced reduction in ERK phosphorylation. Similar results were obtained with human corneal epithelial cells. In keratinocytes, immunoprecipitation studies revealed that ␤ 2 -AR activation resulted in the rapid association of ␤ 2 -AR with PP2A as well as a 37% increase in association of PP2A with ERK2. Finally, ␤ 2 -AR activation resulted in a rapid and transient 2-fold increase in PP2A activity. Thus, we provide the first evidence that ␤ 2 -AR activation in keratinocytes modulates migration via a novel pathway utilizing PP2A to alter the promigratory signaling cascade. Exploiting this pathway may result in novel therapeutic approaches for control of epithelial cell migration.When skin is wounded, keratinocytes within the epidermis must migrate from the wound edges to re-epithelialize the wound surface (1). One of the best characterized mediators of keratinocyte migration is the epidermal growth factor receptor (EGFR) 1 (2-4). The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, are activated upon EGF binding to its cognate receptor (reviewed in Ref. 5). They are activated by phosphorylation on both conserved threonine and tyrosine residues and inactivated upon dephosphorylation by dual specificity phosphatases, tyrosine phosphatases, and serine/threonine-specific phosphatases (5-9). The activation of ERK is tightly controlled. The MAP kinase pathway is usually only transiently activated, and its constitutive activation is sufficient to cause the oncogenic transformation of some cell types (10). Inhibiting ERK phosphorylation and activation prevents growth factor-induced keratinocyte migration (11), demonstrating the pivotal role of ERK in the keratinocyte promigratory signaling pathways (11-13).In addition to the EGFR, keratinocytes also express high levels of the G-protein-coupled receptor, the ␤ 2 -adrenergic receptor, (␤ 2 -AR) (14, 15), whose functional role in the epidermis has not yet been elucidated. Keratinocytes do not express the ␤ 1 -AR (16). Earlier studies have investigated the role of ␤-ARs in epithelial wound healing and migration, but the results have been paradoxical. ␤-Antagonists have been reported to either delay (17, 18) or enhance (19) corneal epithelial wound healing, and ␤-agon...

The Rac1 hypervariable region in targeting and signaling

“…Moreover, several studies have confirmed a role for PP2A in the control of cell migration. 123,124 Finally, proteomic analyses have identified SET in complex with a host of cellular proteins including the Rac/Cdc42 GEF βPIX and the Rac/Cdc42 effector PAK1, 125 actin and dynamin-2 119 and a β1-integrin-filamin complex. 126 A recent study by Switzer et al 127 showed that a SET-binding peptide (COG112) does not only block the Rac1-SET interaction, but also reduces EGF-induced Rac1 activation and serum-induced cell migration and invasion.…”

β-Adrenergic Receptor Antagonists Accelerate Skin Wound Healing

“…Finally, we demonstrate that keratinocytes express protein for two key enzymes in the catecholamine synthesis cascade localized to cytoplasmic granules and measure epinephrine in keratinocyte extracts. We believe that this is the first demonstration that ␤-AR antagonists can accelerate human skin wound re-epithelialization, and we hypothesize that the mechanism of action is via blockade of an endogenous autocrine ␤2-AR network that slows migration and delays wound healing (33,34).…”

“…Upon mechanical injury of confluent keratinocyte cultures (42) or Madin-Darby canine kidney cell cultures (43), ERK is activated by and is required for wound repair in confluent rat keratinocyte cultures (63) and in human epidermis (64). Previously, we have demonstrated that phospho-ERK is localized at the lamellipodial edge in migrating keratinocytes (33) and that ␤-AR agonists decrease ERK phosphorylation and prevent its localization to the lamellipodia via protein phosphatase 2A-dependent mechanisms (33,34). Although the function of phospho-ERK at the lamellipodial edge is unknown, it is possible that direct interactions between ERK and ␤ integrins (65) or paxillin (66) take place, suggesting an important role for ERK in integrating cell adhesion and receptor-mediated signaling in the control of cell migration.…”

“…Cultures-To determine the effect of ␤-AR antagonists on keratinocyte migration, we initially measured the ability of keratinocytes to heal a "scratch wound" within a confluent sheet of cells (34). A ␤-AR antagonist, at a concentration specific for ␤2-AR (10 nM) (27), doubles the rate of scratch wound healing.…”

The Proto-oncogene SET Interacts with Muscarinic Receptors and Attenuates Receptor Signaling

“…PP2A is one of the major phosphatases involved in GPCR dephosphorylation (25). It binds directly to intracellular domains of the chemokine receptor CXCR2 (33) and the metabotropic glutamate receptor 5 (34) and co-immunoprecipitates with the ␤2-adrenergic receptor (35). In HEK293 cells stably expressing the CXCR2 receptor, inhibition of PP2A by okadaic acid increases basal phosphorylation of the CXCR2 receptor and reduces the increase in intracellular calcium concentration after a short-term stimulation with the agonist CXCL8 (33).…”