Protein kinase C-dependent phosphorylation of the beta-subunit of the voltage-sensitive potassium channels (Kvbeta2).
The K(v)beta proteins are members of the aldo-keto reductase (AKR) superfamily that interact with the cytoplasmic face of the pore-forming alpha-subunits of the voltage-sensitive K(+) channels. The physiological functions of K(v)beta are unclear, although some members of the K(v)beta 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(v)beta proteins. The K(v)beta2 displays high-affinity NADPH binding, although no catalytic activity for this protein has been reported. Recent studies show that K(v)beta2 is constitutively associated with protein kinase C (PKC) zeta via the zeta-interacting protein (ZIP). The primary structure of K(v)beta displays 25 PKC consensus phosphorylation sites. We report that incubation of recombinant K(v)beta2 with PKCalpha 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(v)beta2 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 beta-alpha interactions or the binding of NADP(H) to the conserved AKR domain of the K(v)beta proteins.
Liu
Si-Qi
SQ
Division of Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, USA.
Yin
Jianning
J
Bhatnagar
Aruni
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C