Physiological Sciences GIDP
Experimental Biology Meeting
San Diego, California
April 2-6, 2016
Serine 275 and serine 328 in the carboxyl-terminus regulates phosphorylation-dependent anti-proliferative properties of connexin 37
Authors: Nicole L. Jacobsen, Samantha Taylor, Tasha K. Pontifex, and Janis M. Burt Physiological Sciences GIDP, Department of Physiology, University of Arizona, Tucson, AZ
Connexin 37 (Cx37), but not Cx40 or Cx43, suppresses the proliferation of rat insulinoma (Rin) cells, an effect requiring both its channel function and regulation mediated by the carboxyl-terminus (CT). Additionally, data from cells expressing alanine or aspartate substituted forms of Cx37 suggest that phosphorylation at specific serine residues (275, 285, 302, 319, 321,325, 328) within the CT directs cell survival and proliferation; “dephosphorylated” Cx37-CT (use of alanine substitution) results in cell cycle arrest while “phosphorylation” of the Cx37-CT (aspartate substitution) induces cell death. Interestingly, aspartate substitution at only serines 275, 319, 328 (S3D3) neither limits Rin cell growth nor induces cell death. Rather, there is no effect on the rapid Rin cell proliferation despite its expression. As the first identified full-length form of Cx37 that retains channel functionality while supporting cell proliferation, we investigated whether phosphorylation at a single site would be sufficient to control cell cycle progression. Single point mutations with alanine and aspartate were made at each of the three sites and subsequently expressed in Rin cells. Aspartate substitution at serine 275, a putative MAPK site, induces cell death while alanine substitution does not have a significant impact on the anti-proliferative effects of Cx37-WT. Mutation of serine 319 to either an alanine or aspartate no longer limits Rin cell proliferation, suggesting that this site may not be critical to phenotype determination. Finally, expression of Cx37-S328A (alanine substitution at a putative PKC site) results in cell death but Cx37-S328D supports proliferation. Together, these data may suggest that serine 275 and 328 cooperate in modulating cell proliferation in a phosphorylation-dependent manner.
Supported by: R01HL058732 and T32HL007249.
Abstract for Lay Audience
Our research is aimed at understanding how gap junction channels regulate the growth of vascular cells. In particular, connexin 37 (Cx37), a gap junction protein expressed in the endothelial layer of arterial blood vessels appears to be critically important in the development of new blood vessels in addition to the remodeling of existing vessels. In a setting of ischemic injury, as seen in atherosclerotic peripheral artery disease (PAD), Cx37 limits the potential for recovery due to a decrease in the number of vessels carrying blood and consequently a reduction in total blood flow to the injured tissue. Therefore, discovering the mechanism of Cx37-mediated growth regulation could prove beneficial in providing insights into possible therapeutic strategies in the treatment of PAD. We discovered that when expressed in highly proliferative cancer cells, Cx37 significantly slows the proliferation of the cells. Also, protein modification via phosphorylation in a regulatory region of the Cx37 protein modulates the growth suppressive behavior. In the current study, we targeted specific amino acids in the region of interest with the hopes of identifying a single phosphorylation event responsible for the growth suppressive phenotype. Using site directed mutagenesis, we created multiple “phosphorylated” and “dephosphorylated” forms of Cx37 using aspartate and alanine substitutions, respectively. By using an aspartate for serine substitution, it can mimic a phosphorylation event at that site due to similarity in charge. In contrast, an alanine substitution at the residue will prevent phosphorylation from occurring at that specific amino acid as a result of the absence of the hydroxyl group. We found that aspartate substitution at serine 275, serine 319, and serine 328 altogether alleviated Cx37’s control over cell cycle progression implicating at least one, if not all, sites in the growth suppression mechanism by Cx37. Next, we investigated whether phosphorylation at only a single site would be sufficient to control Cx37- mediated cell cycle progression. Single point mutations with aspartate and alanine were made at each of the three sites individually. The resulting data may suggest that serine 275 and serine 328, but not serine 319, cooperate in modulating cell proliferation in a phosphorylation-dependent manner. Based on the results from the current study, it can be suggested that promoting the phosphorylated/growth permissive form of Cx37 would be advantageous in the treatment of PAD. By encouraging growth of new blood vessels following ischemia, more blood would be able to perfuse the peripheral tissue and result in a quicker recovery due to quicker return of critical nutrients and signaling molecules.