Title: C-terminal lysines modulate Connexin32 turnover and its ability to suppress growth of Neuro-2a cell cultures
Description: The extent of gap junction (GJ)-mediated coupling can be modulated through GJ channel gating. However, the amount of connexin protein available for incorporation into GJ, efficiency of channel assembly, trafficking to the cell surface, and disassembly also contribute to the regulation of cell-cell communication. In addition to their function in GJ, connexins also regulate a variety of physiological processes by forming hemichannels that are involved in paracrine signaling (Sanchez, Orellana et al. 2009) and through interactions with other proteins in the cytoplasm(Francis, Xu et al. 2011) and at the plasma membrane(Fowler, Akins et al. 2013). These other connexin functions are also likely to be influenced by the channel assembly dynamics, trafficking, and fast turnover of connexin proteins. The aim of this work was to determine if post-translational modifications, such as lysine acetylation, regulate connexin function through the fine-tuning of protein turnover or some other aspect of GJ dynamics.

We chose to focus specifically on Cx32 for several reasons. Cx32 is an important regulator of neuronal myelination and loss of Cx32 GJ function is a common cause of the demyelinating neuropathy, Charcot-Marie-Tooth disease. Most studies addressing post-translational modifications of connexins focus on Cx43, which shares little sequence homology with Cx32 in the domains that are most-often subject to post-translational modification. We surmised that our results could be compared to what is known about Cx43 in order to determine if shared post-translational modifications regulate evolutionarily divergent connexins in similar ways. Here we show that Cx32 is an acetylated protein and that acetylated Cx32 is found in the cytoplasm and in the plasma membrane, where it is incorporated into GJ. For many proteins, acetylation has been implicated in pathways that modulate protein turnover(Caron, Boyault et al. 2005), thus we tested whether acetylation could regulate Cx32 protein level, resulting in the modulation of Cx32 functions.

Our results demonstrate that acetylation is a positive regulator of Cx32 protein level, which increases the amount of Cx32 at the cell surface. Inhibition of the cytoplasmic deacetylase, HDAC6, results in hyperacetylation and accumulation of Cx32, which is dependent upon cytoplasmic C-terminal lysines. Mutational analysis revealed that these C-terminal lysines influence the ubiquitination and turnover rate of Cx32 protein. Comparison of the subcellular localization of WT Cx32 to that of mutants that either abolish acetylation sites while maintaining the original amino acid charge (K → R) or mimic constitutive acetylation (K → Q) suggests that acetylation does not simply alter lysine occupancy, thus preventing Cx32 ubiquitination and subsequent turnover. Instead, it seems likely that acetylation modulates protein-protein interactions that influence the amount of Cx32 in the plasma membrane and the role of Cx32 as a regulator of growth of cell cultures. K → Q Cx32 accumulates at the cell-surface more than WT Cx32, while K → R behavior resembles that of WT.

Further, K → Q Cx32 suppresses the expansion of N2a cell cultures, whereas WT and K → R Cx32 do not. Interestingly, none of the mutations resulted in detectable alterations of cell-cell communication, suggesting that the suppression of cell culture growth observed when cells expressed the K → Q mutant may be independent of cell-cell communication. These results suggest that Cx32 acetylation is a positive regulator of Cx32 mediated suppression of proliferation or enhancement of pro-apoptotic signaling and provide rationale for future studies to determine which protein-protein interactions are modulated through Cx32 acetylation.