Obscurin regulates ankyrin macromolecular complex formation
Graphical abstract
Introduction
Cardiomyocyte contraction is a tightly regulated process that involves the coordinated assembly of cytoskeletal and scaffolding proteins of the sarcomere and internal organelles that regulate calcium cycling. Adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play pivotal roles in guiding the incorporation of myofilament and accessory proteins into striated structures and contribute to the alignment of the sarcoplasmic reticulum (SR) and transverse tubules (T-tubules). How these cytoskeletal and cytoskeletal-associated proteins contribute to the subcellular organization of the cardiomyocyte provides valuable information about normal cardiomyocyte function as well as dysfunction in disease.
Obscurins, encoded by the gene OBSCN, are a family of sarcomeric structural and signaling proteins localized to Z-lines and M-lines of myocytes [[1], [2], [3]]. Several mutations in OBSCN have been linked to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), left ventricular noncompaction cardiomyopathy (LVNC), heart failure, and skeletal myopathies [[4], [5], [6], [7]]. The differential subcellular localization of obscurins combined with its numerous modular adhesion and signaling motifs enable it to play pivotal roles in the organization of the sarcomere and maintenance of A-bands and M-lines [1,[8], [9], [10], [11], [12], [13]], in the organization of costameres to withstand stress and transmit contractile force across the sarcolemma [14], and in the organization and cytoskeletal anchoring of the SR membranes [[15], [16], [17], [18], [19]]. One difference between the obscurins found at the M-line, or obscurin-A, versus the Z-line is the presence of two independent ankyrin binding domains in the carboxyl terminus of obscurin-A [20].
Ankyrins are versatile adaptor proteins that facilitate the recruitment and maintenance of protein complexes to their distinct subcellular domains by tethering the integral membrane proteins and signaling molecules to the underlying β-spectrin cytoskeleton. Three genes encode ankyrin proteins (ANK1: ankyrin-R or AnkR, ANK2: ankyrin-B or AnkB, ANK3: ankyrin-G or AnkG) and alternative splicing of these genes yields several modular isoforms [21]. Mutations in ankyrins are linked to several arrhythmias including atrial fibrillation, sinus node dysfunction, long QT syndrome (LQTS), ventricular tachycardia, and Brugada syndrome [[22], [23], [24], [25]]. Furthermore, recent studies show that ankyrin dysfunction has also been associated with arrhythmogenic cardiomyopathy (ACM), DCM, and HCM [[26], [27], [28]].
A canonical ankyrin is composed of four functional domains: the membrane-binding domain (MBD) that binds integral membrane proteins, spectrin-binding domain (SBD) that tethers protein complexes to the cytoskeleton, death domain (DD), and C-terminal domain (CTD). The DD and CTD comprise a regulatory domain because an intramolecular interaction with the MBD regulates MBD and SBD binding activities [29,30]. While the MBD, SBD, and DD share amino acid homology, the CTD shows sequence diversity, conferring unique regulatory mechanisms to each ankyrin protein [30,31]. Within the CTD of many muscle-specific ankyrin isoforms, there reside obscurin binding domains (OBD) that mediate interaction with obscurin [19,[32], [33], [34], [35]].
Many mechanisms contribute to the appropriate subcellular localization of ankyrin protein complexes including interactions with cell adhesion molecules, cytoskeletal proteins, and/or large scaffolding proteins. This paper is the first to investigate whether two independent ankyrin binding domains in the carboxyl terminus of obscurin-A can interact with two different ankyrins in vitro and in cells. Specifically, we examined obscurin interaction with sAnk1.5 and AnkG107. Small Ank1.5 is a ~ 20 kD muscle-specific ankyrin-R isoform, which lacks the MBD and SBD but has a unique transmembrane domain tethering it to the sarcoplasmic reticulum [17,[36], [37], [38]]. Obscurin interaction with sAnk1.5 is important to maintain the longitudinal sarcoplasmic reticulum (lSR) across the contractile apparatus to facilitate uniform calcium release and re-uptake into the SR [18,19,39,40]. AnkG107 is a muscle-specific ankyrin-G isoform that lacks the MBD but has two putative OBDs in its C-terminal domain. While it has been shown that AnkG107 interacts with obscurin by yeast two-hybrid analysis, it has also been reported that the OBDs mediate interaction with plectin-1 and filamin-C [41].
This paper is the first to demonstrate that obscurin can interact with two different ankyrins at the same time. This observation is validated by in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis. Specifically, we demonstrate that sAnk1.5 and AnkG107, which do not interact with each other, are brought together in a complex via the two ankyrin-binding domains in obscurin. We also find that AnkG107 binding obscurin is principally mediated by the first OBD, and that the OBDs do not bind filamin-C and display minimal binding to plectin-1 compared to obscurin. In addition, we show that AnkG107 recruits β1-spectrin to a complex of obscurin and sAnk1.5. Finally, we demonstrate that virally-expressed sAnk1.5 and AnkG107-CTD localize to the M-lines of cardiomyocytes and this localization is disrupted when the obscurin-binding domains are mutated. Altogether, this work characterizes a novel mechanism by which the large scaffolding protein obscurin coalesces two distinct ankyrin protein complexes to the same domain through interactions with its independent ankyrin-binding domains.
Section snippets
Plasmids and antibodies
Fig. 1 contains a diagram of all constructs used in experiments for this paper. Human sAnk1.5 (AF005213, amino acids 29–155) was subcloned in frame with various carboxyl-terminal epitope tags including FLAG, HA, YFP, CFP, and GFP. Human obscurin (NM_052843, amino acids 6148–6460) was subcloned in frame with an amino-terminal GST epitope tag or with various carboxyl-terminal epitope tags including HA, CFP, and GST. To generate ankyrin-binding domain mutants of obscurin (ΔABD1, ΔABD2, ΔABD1&2),
AnkG107/130-CTD binds obscurin
Previous studies have shown that skeletal muscles express different ankyrin-G isoforms including AnkG107 and AnkG130 [[44], [45], [46], [47]]. The mRNA transcripts encoding these isoforms have an alternative start site in Ank3 exon 25 and include the muscle-specific exons 46–49 [44,[46], [47], [48]]. As a result, these isoforms are similar in that they lack membrane-binding domains but express two obscurin-binding domains in their C-termini (Fig. 2A). In contrast, they are different in that
Discussion
There are many mechanisms that regulate ankyrin-mediated protein complex formation, of which the predominant mechanism is the generation of unique alternatively spliced ankyrin isoforms that interact with distinct cohorts of proteins, targeting these complexes to specific subcellular domains. Several studies have shown that alternative splicing of the three ANK genes results in various tissue-specific, modular isoforms that bind discrete sets of proteins [19,29,[34], [35], [36], [37],44,[46],
Accession codes
sAnk1.5 AF005213 (NCBI) ankG107 AJ428573 (GenBank) obscurin NM_052843 (NCBI) sarcolipin Q6SLE7.1 (Uniprot) plectin-1 NM_011117 (NCBI) filamin-C NM_001081185 (NCBI) β1-spectrin J05500.1 (GenBank)
Author contributions
JS and GY performed the experiments. JS and SRC wrote the paper.
Funding
This work was supported by the American Heart Association (16GRNT30410011).
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
Fluorescence microscopy and image analysis was performed at the Nikon Center of Excellence - Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology at McGovern Medical School, UTHealth Houston.
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