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

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 (ezrinradixin-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 PKAinduced phosphorylation. In vitro [␥-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 growthsuppressive 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.

VprBP targets Merlin to the Roc1-Cul4A-DDB1 E3 ligase complex for degradation

“…Phosphorylation of Merlin at Ser518 was previously reported to play an important role in regulating Merlin tumor suppressor activity (Shaw et al, 2001;Alfthan et al, 2004;Jin et al, 2006a). Interestingly, the interaction between Merlin and VprBP is independent of Ser518 phosphorylation of Merlin (data not shown), suggesting that the Merlin-VprBP interaction might represent a new means for Merlin regulation in vivo.…”

“…Phosphorylation of a C-terminal serine (S518) by p21-activated kinase (PAK)-or cAMP-dependent protein kinase A weakens Merlin's self-association and is believed to inactivate its growth-suppressing activity. On the other hand, the phosphatase MYPT-1-PP1-d activates the tumor suppression activity of Merlin through S518 dephosphorylation, which subsequently inhibits the Ras/ERK (extracellular signal-regulated kinase) pathway (Shaw et al, 2001;Alfthan et al, 2004;Jin et al, 2006a). In addition, Merlin can also be regulated at the protein level by growth arrest stimuli.…”

Serine 518 phosphorylation modulates merlin intramolecular association and binding to critical effectors important for NF2 growth suppression

“…In this report, we demonstrate that Ser518 phosphorylation results in impaired merlin Nterm/C-term folding as well as altered interactions with critical merlin-associated proteins. Recently, Alfthan et al (2004) demonstrated that protein kinase-A (PKA) induces merlin phosphorylation on both N-and C-terminal residues in vitro and in vivo Like PAK, PKA can phosphorylate merlin at Ser518, but unlike PAK, also results in merlin phosphorylation on Ser66 in the N-terminal domain. In their study, they show that merlin can be phosphorylated by PKA in cells, in which PAK activity was suppressed, indicating that these two kinases likely function independently.…”

Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

“…This ezrin phosphorylation requires upstream p38 MAP kinase activation but is unlikely to be mediated directly by p38 MAP kinase, because ezrin threonine 567 lacks the proline residues that are typically present within p38 MAP kinase targets. Rho kinase, PKA, and some PKC isoforms have been reported to phosphorylate ezrin or other ERM-related proteins (9,11,12,43). However, rho kinase (27) or PKA inhibition does not prevent NHE3 activation after initiation of Na ϩ -glucose cotransport, and both PKA and PKC activation are well known to reduce NHE3 activity and surface expression (30,44).…”