Connexin43 phosphorylation: structural changes and biological effects - PubMed (original) (raw)

Review

Connexin43 phosphorylation: structural changes and biological effects

Joell L Solan et al. Biochem J. 2009.

Abstract

Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, co-ordinated contraction of excitable cells, tissue homoeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin 'life cycle', including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1-5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. The present review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology, with an emphasis on events occurring in heart and skin.

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Figures

Figure 1

Figure 1. Phosphorylation and assembly of Cx43 containing gap junctions

(A) Association of the SDS-PAGE migration of Cx43 in homeostatic (CON) and TPA treated cells with the sites of phosphorylation and a schematic diagram representing channel activity. The cylindrical channels are green to represent promoting communication and red to represent inhibition or closure. The green cross hatch represents an assembling channel and the red a channel with reduced conductance. (B) Gap junction assembly is denoted by cross hatch green channels assembling into solid green communicating channels and red channels represent those that will be internalized and degraded.

Figure 2

Figure 2. Model of the Cx43 C-terminus based on NMR structural studies

(A) Cartoon of a possible structure for the Cx43 C-terminus (amino acids 252−382) with known regulatory sites/binding sequences marked in different colors produced using the PyMOL Molecular Graphics System (

http://www.pymol.org

). (B and C) Space filled models of amino acids 260−292 and 360−382, respectively. Circles mark residues which show resonance peak shifts in NMR spectra in response to SH3 binding (B) or S365D substitution (C). Blue circles mark residues that shift in response binding of the src SH3 domain (B and C). Green circles mark residues that are shifted in a S365D mutant as compared to wild type (B). In (C) dashed lines represent the only residues shown that do not shift due to ZO-1 binding (pink) or the S365D mutation (yellow).

Figure 3

Figure 3. Connexin localization and phosphorylation change dramatically during the cell cycle

Confluent cells in G0/G1 are very efficient at trafficking and assembling (green arrow) most of the Cx43 into Triton X-100 insoluble gap junctions, shown in green. ZO-1 and ZO-2 are colocalized at these plaques. As cells progress into S phase assembly of gap junctions becomes less efficient and Cx43 is found both in gap junction plaques and in cytoplasmic vesicles (green). Association with ZO-1 is decreased while ZO-2 interaction is maintained. PKC-mediated phosphorylation of Cx43 on S368 and S262 (transient?) begins occurring as cells approach S phase. As cells enter mitosis, gap junction communication ceases (red) and Cx43 is found predominantly in clusters of vesicles in the cytoplasm (green). Cx43 becomes increasingly phosphorylated on S368 and S255 and exhibits a migration shift by SDS-PAGE. Cells are able to quickly resume communication upon cytokinesis, likely due to plasma membrane connexon pools.

Figure 4

Figure 4. Schematic diagram of connexin expression in normal and wounded human skin

Cx43 expression (denoted in green) in unwounded skin is mainly in the upper more differentiated layers. Upon wounding Cx43 expression drops very near the wound and redistributes to lower layers in the cells several cells distant from the wound. The basal cells near the wound express Cx43 that is highly phosphorylated at S368 (indicated by orange) forming a distinct communication compartment.

Figure 5

Figure 5. Schematic diagram of changes in connexin expression in heart under ischemic conditions alone or with preconditioning

Normally cardiac myocytes express high levels of Cx43 at the intercalated disc (ID) and gap junctions are in their open state, shown in green. Under ischemic conditions there is a loss of gap junctions at the intercalated disc as Cx43 moves to the lateral edges of the myocyte, shown in red. Some Cx43 is retained at the intercalated disc, but it becomes phosphorylated on S368 (indicated by orange). In addition, PKCε translocates from the cytoplasm to the plasma membrane and may be involved in Cx43 phosphorylation on S368. With ischemic preconditioning, gap junctions are retained at the intercalated disc with no subsequent S368 phosphorylation. PKCε is necessary for this retention and is found at the plasma membrane. In addition, increased Cx43 may be found in the mitochondria.

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