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Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

Human proliferation-associated protein p120 has previously been shown to localize to the nucleolus, and several functional domains of p120 have been elucidated. By using a nitrocellulose filter binding assay and a Northwestern blotting procedure this study shows that recombinant p120 binds to an rRNA fragment in vitro with a dissociation constant of 4 nM. The specific RNA-binding region of p120 (residues 1–57) was identified with glutathione S-transferase-fused p120 deletion constructs and Northwestern blotting procedures. This RNA-binding region of p120, which includes the nucleolar localization signal of p120, is similar to the arginine-rich RNA-binding regions found in other RNA-binding proteins such as HIV Rev and Tat. Experiments in vivo with HeLa cell nucleolar extracts showed that p120 was associated with the 60–80 S pre-ribosomal particles. This association is disrupted by treatment with either RNase A or buffer of high ionic strength. These results suggest that p120 might be involved in rRNA/ribosome maturation, consistent with the role of the yeast homologue Nop2p in rRNA biogenesis.