Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival

Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273⁻317) and end-tail (aa 318⁻333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death

Connexin 37 Growth Suppressive Phenotype is Regulated in a Site-Specific, Phosphorylation-Dependent Manner

Connexins support the coordinated function of virtually all tissues in the body through facilitation of intercellular, transmembrane, and intracellular signaling. Connexin 37 (Cx37), which plays a role in vascular development and remodeling, has growth suppressive properties that depend on its channel function and the presence and phosphorylation status of its carboxylterminus (CT). In settings of injury, Cx37 expression is reduced - a change permissive to vascular repair. Serine 319 (S319) is a high probability (>90%) target for phosphorylation by growth factor-activated kinases, and the phosphorylation status of this site is important to the inflammatory response to injury and atherosclerosis. Here, we substituted S319 with alanine or aspartate, to mimic the dephosphorylated (S319A) and phosphorylated (S319D) states, respectively, and transfected rat insulinoma (Rin) cells with these mutants to elucidate the mechanistic basis for phospho-form specific roles of Cx37-S319 in growth control. Both mutants formed functional gap junction channels, but Cx37-S319A relieved the growth suppressive effect of Cx37, whereas Cx37-S319D did not. Altogether, these data suggest that preventing phosphorylation at S319 is sufficient to relieve the growth suppressive effect of Cx37, but the mechanistic basis for this effect is likely through induced changes in protein-protein interactions

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.National Institutes of Health12 month embargo; first published online 18 June 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Regulation of Cx37 channel and growth-suppressive properties by phosphorylation

Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.National Institutes of Health [HL056732, HL007249, HL131712, GM072631, GM55632, CCSG - CA023074]; American Heart Association [16PRE27500011]12 month embargo; Published online: 1 Oct 2017.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]