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Cx45 regulation by kinases and impact of expression in heart failure
Gaelle Spagnol1, Andrew Trease1, Li Zheng1
1From the Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Abstract:
Phosphorylation plays a crucial role in connexin regulation by modulating gap junction intercellular communication (GJIC), localization, stability, and interactions with signaling proteins. Few kinases are known to phosphorylate Cx45, and their target residues remain unknown. A phosphorylation screen identified several Cx45-targeting kinases activated in heart disease, among which c-Src was found by mass spectroscopy to phosphorylate residues Y324 and Y356. Unlike Cx43, c-Src phosphorylation of Cx45 did not impair GJIC, alter junctional localization, or affect interactions with cytoskeletal proteins β-tubulin, Drebrin, and ZO-1. In LA-25 cells where Cx43 is internalized after temperature sensitive activation of v-Src, expression of Cx45 unexpectedly maintained Cx43 at the plasma membrane. Phospho-specific antibodies helped identify that while Cx43 had a tyrosine phosphorylation pattern favoring turnover, the serine phosphorylation pattern was conducive for GJIC. Furthermore, in a rat model of heart failure, Cx45 was expressed in the ventricle and co-localized with Cx43, leading to altered dye coupling indicative of a shift toward Cx45-like channel permeability. Altogether, our data suggests that in heart failure, c-Src activation on its own would not have an adverse effect on Cx45 function and that aberrant Cx45 expression helps Cx43 transport to and maintain at the intercalated disc. Yet the dominant effect of Cx45 in heteromeric channels could ultimately make Cx45 a key driver of cardiac dysfunction. Finally, the observation that Cx45-mediated coupling remains functional even in the same pathological environment where Cx43-mediated communication is inhibited suggests that kinase regulation of connexins is isoform-specific and not universally predictable.
Insights
This study reveals how c-Src phosphorylates Cx45 in heart disease, impacting connexin function. Aberrant Cx45 expression may contribute to cardiac dysfunction by altering Cx43 localization and channel permeability.
Area of Science:
- Cardiovascular Biology
- Cell Signaling
- Molecular Cardiology
Background:
- Phosphorylation regulates connexin function, including gap junction intercellular communication (GJIC), localization, and stability.
- Specific kinases and phosphorylation sites for connexin 45 (Cx45) are largely unknown, hindering understanding of its role in cardiac disease.
Purpose of the Study:
- To identify kinases that phosphorylate Cx45 and their target residues.
- To investigate the functional consequences of Cx45 phosphorylation by c-Src in the context of heart disease.
- To elucidate the role of Cx45 in Cx43 regulation and cardiac dysfunction.
Main Methods:
- Phosphorylation screening and mass spectrometry to identify Cx45-targeting kinases and residues.
- Cellular assays (LA-25 cells) to assess GJIC, protein localization, and interactions.
- Use of phospho-specific antibodies to analyze connexin phosphorylation patterns.
- In vivo studies using a rat model of heart failure.
Main Results:
- c-Src phosphorylates Cx45 at tyrosine residues Y324 and Y356.
- Unlike Cx43, c-Src phosphorylation of Cx45 does not impair GJIC, junctional localization, or cytoskeletal interactions.
- Cx45 expression maintains Cx43 at the plasma membrane in v-Src-activated LA-25 cells.
- In heart failure, Cx45 is expressed in ventricles, co-localizes with Cx43, and alters dye coupling, suggesting a shift towards Cx45-like channel permeability.
- Cx43 phosphorylation patterns favor turnover (tyrosine) or GJIC (serine).
Conclusions:
- c-Src activation alone does not adversely affect Cx45 function in heart failure.
- Aberrant Cx45 expression aids Cx43 transport and maintenance at the intercalated disc.
- Dominant effects of Cx45 in heteromeric channels may drive cardiac dysfunction.
- Kinase regulation of connexins is isoform-specific, as Cx45-mediated coupling remains functional where Cx43 coupling is inhibited.
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