Abstract
Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.
Highlights
Connexin43, a ubiquitous gap junction protein, is phosphorylated by protein kinase C on serine 368
By activating protein kinase C (PKC) with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes
We first started our experiments with intermolecular FRET Ratio ϭ Average ROI pixel intensity (FRET) (YFP-PKC␦ and Cx43-CFP) to examine the recruitment of PKC␦ to Cx43, we found the interaction of PKC with Cx43 occurred too quickly and too transiently for good spatiotemporal resolution
Summary
A ubiquitous gap junction protein, is phosphorylated by protein kinase C on serine 368. Results: After PKC␦ activation, phospho-Ser-368 Connexin channels segregated into the gap junction center and were subsequently internalized and degraded. Conclusion: PKC␦ phosphorylation triggered internalization and degradation of Connexin channels without dephosphorylation. Significance: Differential phosphorylation events are used to sort and traffic Connexin channels within gap junctions and into the cytoplasm. Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism
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