Connexin37 Phosphorylation Status Determines Different Growth Phenotypes

Abstract

Cellular growth regulation can be modulated by altering the phosphorylation state of Cx37. Mechanistically it remains to be fully elucidated how Cx37 contributes to this regulation by means of: intramolecular binding partners, its function as a hemi-channel or by facilitation of intercellular communication via gap junctions. Herein we sought to examine the effects of phosphorylation by means of phosphomimetic substitutions (serines to aspartates) on proliferation and the possible alterations to the protein channel function by looking at three mutants Cx37-S275,319D, Cx37-S275,328D and Cx37-S275,319,328D expressed in rat insulinoma cells(Rin). Growth curve assays showed that cells with either Cx37-S275,319D or Cx37-S275,328D eliminated the cell death seen in Cx37-WT and previously studied Cx37 proteins with single site phosphomimetic substitutions at either S275D or S328D, as well as allowing unrestrained proliferation when cells were induced to express the protein at higher densities. Rin cells with Cx37-S275,319,328D appear to proliferate at any density without any measurable cell death. There was a reduction in the open probability for large hemi-channel events for all three non-death inducing mutants and an increased probability of hemi-channels being in the closed state for the growth arrested cells expressing Cx37-S275,319D and Cx37-S275,328D. These data suggest that differential phosphorylation could be an important factor modulating hemi-channel behavior either promoting the closed-state, and thereby facilitating growth arrest and/or preventing high current Cx37 hemi-channel openings and thus facilitating Cx37’s capacity to cause cell death.