Merlin Links to the cAMP Neuronal Signaling Pathway by Anchoring the RIβ Subunit of Protein Kinase A

摘要

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIβ is most abundantly expressed in brain. The RIβ knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIβ in learning and memory-related functions. Molecules that interact with or regulate RIβ are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIβ. Merlin and RIβ demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the α-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved α-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIβ and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling. The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIβ is most abundantly expressed in brain. The RIβ knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIβ in learning and memory-related functions. Molecules that interact with or regulate RIβ are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIβ. Merlin and RIβ demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the α-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved α-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIβ and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling. The neurofibromatosis 2 (NF2) 1The abbreviations used are: NF2, neurofibromatosis 2; ERM, ezrinradixin-moesin; PKA, protein kinase A; AKAP, protein kinase A anchoring protein; mAb, monoclonal antibody; GFP, green fluorescent protein; GST, glutathione S-transferase. tumor suppressor protein merlin (schwannomin) is structurally related to ezrin-radixinmoesin (ERM) proteins (1Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Nature. 1993; 363: 515-521Crossref PubMed Scopus (1207) Google Scholar, 2Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1106) Google Scholar), which link the actin-containing cytoskeleton to specific membrane proteins. ERM proteins also interact with cytoplasmic signaling molecules and participate in Rho and PKC signaling (3B