QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 April 2003
doi: 10.1242/jcs.00428

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Solan, J. L. Articles by Lampe, P. D. Search for Related Content PubMed PubMed Citation Articles by Solan, J. L. Articles by Lampe, P. D.

Connexin43 phosphorylation at S368 is acute during S and G₂/M and in response to protein kinase C activation

¹ Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA, and Department of Pathobiology, University of Washington, WA 98195, USA
² Cell Signaling Technology, Beverly, MA 01915, USA
³ Genetics, Cell Biology and Development, University of Minnesota, St Paul, MN 55108, USA

View larger version (92K): [in a new window]   Fig. 1. The p368 antibody reacts with Cx43 only when S368 is present. Shown is an immunoblot of whole cell lysates from HeLa cells transfected with wild-type (wt) Cx43 or Cx43 containing a S368A mutation. Cells were either incubated in the presence (TPA) or absence (CON) of 50 ng/ml TPA for 30 minutes. The immunoblot was probed with either an antibody to the N-terminal region of Cx43 (-Cx43) or the anti-p368 antibody (-p368). Positions of the molecular weight markers are shown on the left.

View larger version (49K): [in a new window]   Fig. 2. The p368 antibody reacts with Cx43 only when it is phosphorylated at S368. Untreated (CON) or TPA-treated cells were lysed in sample buffer or treated with alkaline phosphatase (+AP lanes) prior to SDS-PAGE and immunoblotting. The blot was probed with the p368 antibody (-p368 panel) followed by stripping and reprobing with the Cx43 antibody (-Cx43 panel).

View larger version (63K): [in a new window]   Fig. 3. TPA-treated NRK cells show increased phosphorylation at S368 with no apparent shift in migration in SDS-PAGE. NRK cells were metabolically labeled with [32P]orthophosphate and incubated in the presence (TPA) or absence (CON) of 50 ng/ml TPA for 30 minutes followed by immunoprecipitation of Cx43, blotting to nitrocellulose, and probing the blot first by autoradiography (32P) and then using the anti-p368 antibody (-p368) and ultimately the N-terminal Cx43 antibody (-Cx43).

View larger version (68K): [in a new window]   Fig. 4. The p368 antibody immunoprecipitates more Cx43 from either NRK or CHO cells after TPA treatment. NRK and CHO cells were metabolically labeled with [35S]methionine and either treated with TPA or left untreated (CON) and then lysed and immunoprecipitated with either the Cx43 antibody (-Cx43 lanes) or the p368 antibody (-p368 lanes).

View larger version (104K): [in a new window]   Fig. 5. NRK and CHO cells both show large increases in phosphorylation at S368 upon TPA treatment but the resulting Cx43 mobilities are very different. Cells were either incubated in the presence of no drugs (CON), 50 ng/ml TPA for 30 minutes (TPA) or 5 µg/ml brefeldin A for 4 hours (BFA), and processed for immunoblot and separately probed with the anti-p368 antibody (-p368) and the N-terminal Cx43 (-Cx43).

View larger version (59K): [in a new window]   Fig. 6. The p368 antibody binds to both junctional and cytoplasmic membranes. NRK cells that had been incubated in the presence (TPA) of 50 ng/ml TPA or absence (CON) were processed for immunofluorescence with the anti-p368 (-pS368) and the Cx43IF1 (-Cx43) antibodies (left and center panels) or with the anti-p368 antibody plus the immunizing peptide (right panel, -pS368 + peptide).

View larger version (94K): [in a new window]   Fig. 7. The extent of p368 phosphorylation is increased through the cell and is maximal during S and G2/M. Synchronized cells collected at the indicated cell-cycle stage were processed for immunoblotting and probed with antibodies to p368 (-p368), Cx43NT1 (-Cx43), or vinculin (for a loading control). The molecular weight or migration position of the Cx43 is indicated on the right, and the ratio of the extent of p368 to Cx43NT1 antibody labeling is shown on the bottom line.

View larger version (77K): [in a new window]   Fig. 8. G0- and S-phase cells show different Cx43 cellular distributions and different abilities to transfer fluorescent dyes in established and junctional assembly assays. (A) G0 cells show extensive junctional Cx43 immunostaining. (B) S-phase cells show extensive junctional and cytoplasmic membrane staining. (C) The number of cells that receive either Alexa488 (A488) or Alexa594 (A594) from the injected cell is quantitated for cells in established G0- or S-phase cultures (mean±s.d.). (D) Cells in G0 or S were assayed for the ability to assemble gap junctions and transfer calcein (quantitated as the number of transfers per the total number of interfaces between a calcein-loaded and a recipient cell, mean±s.d.).