Growth Suppression Mechanism of Connexin 37 in Response to Phosphorylation State of the Carboxy Terminus

Abstract

Connexin 37 (Cx37), profoundly suppresses the growth of rat insulinoma (Rin) cells and requires a functional channel. To determine if the CT is necessary for Cx37-mediated growth suppression, a series of deletions and mutations affecting the availability of putative phosphorylation sites in the region of 273-333 of Cx37 were generated. Truncation at P273 alleviated the growth suppressive effect of Cx37 while retaining channel functionality. Interestingly, substate behavior is lost in iRin37-273tr. Based on the observation of run-down behavior in Cx37-wt channels consistent with dialysis of PKC, TPA and BIM treatment of Cx37- wt channels revealed that PKC activation generates a higher incidence of high conductance channels whereas BIM treatment results in the converse. Exploiting this observation, a S-Ax3 mutation targeting known MAPK and PKC sites aligned from Cx43, was generated. However, channels are able to reside in the sub-conductance state and cells are anti-proliferative. Four additional serine to alanine mutations at putative phosphorylation sites (>90%) were made to generate an S7A mutation and did not dispel the growth suppressive effect of Cx37. A phosphomimetic S7D mutation appears to induce cell death. Based on these data, it appears that substate behavior regulated by the CT is indicative growth suppression by Cx37.