Cyclic AMP-dependent Protein Kinase Phosphorylates Merlin at Serine 518 Independently of p21-activated Kinase and Promotes Merlin-Ezrin Heterodimerization

Mikaela Grönholm,Leena Heiska,G Herma Renkema,Olli Carpén,Kaija Alfthan

doi:10.1074/jbc.m313916200

Mikaela Grönholm, Leena Heiska + Show 3 more

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https://doi.org/10.1074/jbc.m313916200

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Abstract

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrin-radixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKA-induced phosphorylation. In vitro [gamma-(33)P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growth-suppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.

Highlights

Mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene encoding merlin induce the development of tumors of the nervous system
We have recently shown that merlin binds to a regulatory subunit (RI␤) of protein kinase A (PKA) and may function as an AKAP for RI␤-containing PKA both in the central nervous system and in cultured neuronal cells [24]
In this work we studied whether merlin would serve as a substrate for PKA

Summary

Introduction

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growthsuppressive to a growth-permissive state. Like the ERM proteins, merlin can be divided into three apparent structural domains: the globular N-terminal FERM domain, an extended ␣-helical region, and a short C-terminal domain Both inter- and intramolecular associations can regulate the functions of merlin and its interactions with other proteins (4 – 6). Recent data provide evidence that both phosphorylation of a conserved threonine residue (Thr-567 in ezrin, Thr-564 in radixin, Thr-558 in moesin) in the C terminus and binding of phospholipids regulate the activity of the ERM proteins [7]. Cyclic AMP (cAMP) regulates a number of key cellular processes such as cell growth and differentiation, gene transcrip-

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