AKAP15 Anchors cAMP-dependent Protein Kinase to Brain Sodium Channels

Victoria C Tibbs,Peter C Gray,William A Catterall,Brian J Murphy

doi:10.1074/jbc.273.40.25783

Victoria C Tibbs, Peter C Gray + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.40.25783

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Abstract

The voltage-sensitive sodium channel is regulated by cAMP-dependent protein kinase (PKA) phosphorylation. Using purified preparations of rat brain sodium channels, we have shown that the alpha subunit was phosphorylated by a co-purifying protein kinase. The co-purifying kinase was stimulated by cAMP and phosphorylated PKA substrate peptides. Both the regulatory and catalytic subunits of PKA were detected by immunoblotting in purified sodium channel preparations. Bound PKA was immunoprecipitated with anti-SP19 antibodies directed against the sodium channel alpha subunit. PKA bound to sodium channels phosphorylated the sodium channel alpha subunit on the same four serine residues as observed with exogenously added PKA, indicating that association with the sodium channel does not restrict the sites of phosphorylation. Analysis of proteins with high affinity for the type II alpha regulatory subunit of PKA in a gel overlay assay identified a 15-kDa cAMP-dependent protein kinase-anchoring protein (AKAP) in these preparations. Determination of its amino acid sequence by mass spectrometry revealed two peptides identical to AKAP15, a recently described AKAP that targets PKA to skeletal muscle calcium channels. The co-purifying AKAP was also immunoreactive with antibodies generated against AKAP15, and antibodies directed against AKAP15 co-immunoprecipitated the sodium channel. Our results indicate that PKA is bound to brain sodium channels through interaction with AKAP15. Association of AKAP15 with both skeletal muscle calcium channels and brain sodium channels suggests that it may have broad specificity in targeting PKA to ion channels for regulation.

Highlights

§ Present address: Neurocrine Biosciences Inc., San Diego, CA 92121. 1 The abbreviations used are: PKA, cAMP-dependent protein kinase; RII, type II regulatory subunit of PKA; a 15-kDa cAMP-dependent protein kinase-anchoring protein (AKAP), cAMP-dependent protein [9], and in intact neurons or transfected mammalian cells expressing the type IIA sodium channel [8, 10] on four serine residues in the intracellular loop connecting homologous domains I and II of the ␣ subunit [8]
A single major high molecular mass phosphoprotein of ϳ250 kDa was detected (Fig. 1, first lane). This protein was phosphorylated by exogenous PKA, and it was recognized in immunoblots by the anti-SP19 antibody [32] against the loop connecting domains III and IV of the sodium channel ␣ subunit
These results show that the endogenous protein kinase phosphorylates the sodium channel ␣ subunit

Summary

Introduction

§ Present address: Neurocrine Biosciences Inc., San Diego, CA 92121. 1 The abbreviations used are: PKA, cAMP-dependent protein kinase; RII, type II regulatory subunit of PKA; AKAP, cAMP-dependent protein [9], and in intact neurons or transfected mammalian cells expressing the type IIA sodium channel [8, 10] on four serine residues in the intracellular loop connecting homologous domains I and II of the ␣ subunit [8]. Previous studies have shown that partially purified rat brain sodium channel preparations contain a cAMP-stimulatable protein kinase that phosphorylates the ␣ subunit [6].

Results

Conclusion