Desmosomes (DSMs) are intercellular adhesive junctions, which provide structural integrity to tissues that experience mechanical stress such as the heart. Inherited mutations in DSM proteins, such as desmoplakin (DP), result in lethal cardiac disease. DP is a cytoskeletal linker protein that tethers the intermediate filament (IF) network to sites of cell-cell contact. DP-IF binding is tightly regulated to allow dynamic processes such as DSM assembly to occur, which require looser DP-IF interactions. The DP C-tail tunes DP-IF binding where inhibiting phosphorylation in the C-tail leads to misregulated DP-IF interactions. A point mutation within the DP C-tail has been identified in ARVC raising the possibility that interfering with the regulatory C-tail represents an underlying molecular mechanism contributing to cardiac disease. Preliminary results using mass spectrometry (MS) approaches identified six novel phosphorylation sites all of which fall within consensus motifs for GSK3 kinase cascades. GSK3 inhibition led to enhanced DP-IF interactions where DP is hung up on IF and delays DP incorporation into DSMs. Additionally, MS identified the presence of a novel modification, arginine methylation, at four sites including cardiocutaneous mutation site, arginine 2834. Inhibition of protein arginine methyltransferases (PRMTs), the enzymes that catalyze methylation, phenocopy phospho-deficient DP’s enhanced association with IF suggesting that phosphorylation and methylation post-translational modifications (PTMs) cooperate to mediate DP-IF interactions. Sequence analyses of DP PTM sites reveal the presence of putative arginine-claw conformations within the C-tail, a compact protein fold characterized by the formation of salt-bridges between phospho-serines and arginines. I hypothesize that PTMs induce structural alterations of the C-tail to recruit enzymes that modulate DP-IF interactions during the establishment of intercellular junctions. I propose further that interference with this regulatory machinery contributes to the pathogenesis of disease. Aim 1 will focus on the role of PTMs in regulating DP conformation and function in normal and disease states. Aim 2 will focus on the role of methylation and PRMTs in modulating DP function and its functional impact on DP and DSMs. This proposal promises to reveal novel signaling functions of the DSM and new potential targets for intervention in severe cardiac disorders associated with DSM impairment.
|Effective start/end date||7/1/14 → 6/30/16|
- American Heart Association Midwest Affiliate (14PRE20380540)
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