Protein kinase C-dependent phosphorylation of the β-subunit of the voltage-sensitive potassium channels (K vβ2)

Abstract

The K vβ proteins are members of the aldo-keto reductase (AKR) superfamily that interact with the cytoplasmic face of the pore-forming α-subunits of the voltage-sensitive K + channels. The physiological functions of K vβ are unclear, although some members of the K vβ family impart rapid inactivation to non-inactivating K + channels. Structural analyses reveal that the NADPH-binding site as well as the active site architecture and residues of other AKR proteins are conserved in the K vβ proteins. The K vβ2 displays high-affinity NADPH binding, although no catalytic activity for this protein has been reported. Recent studies show that K vβ2 is constitutively associated with protein kinase C (PKC) ζ via the zeta-interacting protein (ZIP). The primary structure of K vβ displays 25 PKC consensus phosphorylation sites. We report that incubation of recombinant K vβ2 with PKCα in vitro leads to rapid phosphorylation of the protein. Stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) also induced the phosphorylation of K vβ2 expressed in COS-7 cells. Deletion of the first 35 amino acids of the variable N-terminus led to a substantial decrease in the overall extent of phosphorylation in vitro, indicating that the N-terminus peptide is the preferred site of PKC phosphorylation. Analysis of the phosphorylated protein by HPLC coupled with an ion-trap electrospray mass spectrometer revealed phosphorylation of Ser-266 located near the NADPH-binding site of the protein. We propose that phosphorylation could affect the N-terminus-mediated β–α interactions or the binding of NADP(H) to the conserved AKR domain of the K vβ proteins.