Doctor of Philosophy (PhD)
Biochemistry & Molecular Biology
Paul L. Sorgen
Connexins are integral membrane proteins that oligomerize to form gap junction channels. Ions and small molecules diffuse intercellularly through these channels, allowing individual cellular events to synchronize into the functional response of an entire organ. Gap junction channels composed of Connexin43 (Cx43) mediate electrical coupling and impulse propagation in the normal working myocardium. In the failing heart, Cx43 remodeling (decreased expression, altered phosphorylation state, loss at intercalated discs, and increased presence at lateral membranes) contributes to rhythm disturbances and contractile dysfunction. While there is considerable information regarding key interactions of Cx43 in the regulation of gap junction channels, unfortunately, the precise mechanisms that lead to remodeling have not been defined, nor have the critical accessory proteins involved been fully characterized.
Several parameters, such as lipophilic compounds, voltage, pH, and phosphorylation influence the function of Cx43. Importantly, Cx43 phosphorylation is a key regulator of gap junction assembly, stability, degradation, channel gating, and selectivity properties. Changes in the level and pattern of Cx43 phosphorylation are commonly observed in both ischemic and nonischemic forms of human heart failure. Therefore, understanding the mechanistic basis of Cx43 phosphorylation will lead to possible points of therapeutic intervention to restore proper gap junction intercellular communication (GJIC) that has been altered due to heart failure. Much progress has been made on how serine phosphorylation regulates Cx43 (23 serine phosphorylation sites; 8 serine kinases); however, the understanding of tyrosine phosphorylation of Cx43 has been limited to residues Y247 and Y265, both known to be phosphorylated by kinases Src and Tyk2.
Because of this lack of knowledge, the objective of my research is to use a multi-disciplinary approach to identify if additional tyrosine phosphorylation site(s) and kinase(s) play a role in altering Cx43 function in cardiac diseases. Our studies identified Cx43 residue Y313 as a novel site targeted for phosphorylation by Src. Src phosphorylation of Cx43 residue Y313 contributes to intracellular localization of Cx43 and inhibiting the Cx43 interaction with the F-actin binding protein Drebrin. We generated a Cx43 Y313 phospho-specific antibody and demonstrated that Cx43 Y313 phosphorylation is increased in diseased human left ventricle. We identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43 carboxyl-terminal (CT) domain by an in vitro tyrosine phosphorylation kinase screen. Pyk2 interacted with Cx43 at the plasma membrane and intracellularly in HeLa cells, and a heart failure rat model showed an increase in Pyk2 activity and interaction with Cx43 in vivo. Activation of Pyk2 was inhibited by the small molecule PF4618433, leading to increased Cx43 GJIC, and preserved GJIC to the normal level when paired with a Src inhibitor. Our data suggested that phosphorylation of Cx43 by Pyk2 is a “second process” that must be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve cardiac function. This information is pivotal if the role of GJIC in normal and diseased states is to be fully understood and utilized for therapeutic benefit in cardiac pathologies.
Zheng, Li, "Defining the Role of Tyrosine Phosphorylation in the Regulation of Connexin43 in Cardiac Diseases" (2019). Theses & Dissertations. 421.
Available for download on Saturday, December 11, 2021