AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

Abstract

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

Introduction

AMP-activated protein kinase (AMPK) is mainly considered as a sensor of cellular and whole body energy homeostasis [1]. AMPK is activated in response to cellular stresses that decrease intracellular ATP levels, with a subsequent rise in AMP:ATP ratio [2]. AMP is not only an allosteric stimulator of AMPK activity but also prevents dephosphorylation of its Thr172 activation loop residue by protein phosphatases [3]. The Peutz-Jeghers protein LKB1, the first identified upstream kinase involved in AMPK activation [4], [5], directly phosphorylates AMPK in most cells. A rise in intracellular Ca²⁺ can also lead to Thr172 phosphorylation/AMPK activation via Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) [6], [7]. Transforming growth factor-β-activated kinase (TAK-1) has also been reported to control AMPK activity [8].

Several groups have demonstrated that AMPK plays a major role downstream of LKB1 to confer cell polarity [9], [10], [11]. For example, myosin regulatory light chain (MLC) phosphorylation, known to be required for both cell polarity and division [12], [13], has been shown to be a downstream target of the LKB1-AMPK pathway involved in controlling cell polarity [11]. Moreover, the abnormal polarity and cell division phenotype observed in an AMPKα null mutant Drosophila could be rescued using a phosphomimetic MLC transgene [11]. However, MLC is not a direct AMPK target [14]. Several protein kinases are thought to be involved in regulating MLC phosphorylation in smooth muscle and in non-muscle cells [15], [16], [17], [18]. These include Rho kinase (ROK) [15], [16]. Specific Rho family GTPases govern distinct cytoskeletal actin organization through different effector proteins [19], [20]. ROK can phosphorylate several cytoskeletal proteins including the myosin phosphatase-targeting subunit-1 (MYPT1) [21]. MYPT-1 binds to both PP1cδ and phosphorylated MLC, targeting PP1cδ to its substrate and resulting in MLC dephosphorylation and subsequent decreased contractility of actomyosin microfilaments. Phosphorylation of MYPT-1 at Thr696 and Thr854 by ROK inhibits myosin phosphatase activity, thereby increasing steady-state levels of MLC phosphorylation [22]. ROK also phosphorylates and activates LIM-kinase, which in turn phosphorylates and inactivates cofilin [23]. Since cofilin can depolymerize actin microfilaments, its inactivation stabilizes F-actin [24]. A key role of the Rho/ROK pathway in hyperosmotic stress-induced cytoskeletal reorganization of MDCK cells has been recently described [25].

Here we report that, in polarized MDCK epithelial cells, AMPK activation by A769662 leads to a dramatic reorganization of cytoskeletal actin. Concomitantly, ROK downstream targets, namely MLC and cofilin, were phosphorylated. These changes resembled those observed in MDCK cells subjected to hyperosmolarity-induced cell shrinkage, but where AMPK activation was not studied [26]. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.