Serine 319 phosphorylation is necessary and sufficient to induce a Cx37 conformation that leads to arrested cell cycling.

Connexin 37 (Cx37; protein product of GJA4) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its C-terminus (CT). In Rin cells, growth is arrested upon induced Cx37 expression and serine 319 (S319) is frequently phosphorylated. Here, we show that preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth-suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced the growth-suppressive properties of Cx37. Like wild-type Cx37 (Cx37-WT), Cx37-S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest versus growth-permissive phenotypes. That the closed state of Cx37-WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.