Multiple Molecular Interactions Implicate the Connectin/Titin N2A Region as a Modulating Scaffold for p94/Calpain 3 Activity in Skeletal Muscle

Abstract

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium

“…2). Studies of ANKRD1 protein interactions have focused on the use of the normally sized protein, or on its N-or C-terminal domain in isolation (Zou et al, 1997;Torrado et al, 2004Torrado et al, , 2005Witt et al, 2005;Hayashi et al, 2008). The present work suggests that ANKRD1 isoforms that lack a part of their C-termini can be present in cardiomyocytes and will require further detailed analysis.…”

The enigmatic role of the ankyrin repeat domain 1 gene in heart development and disease

“…Consistent with these findings, FLAG- (Jeyaseelan et al, 1997) or GFP-tagged (Miller et al, 2003;Zolk et al, 2003) ANKRD1/CARP was localized in both the nucleus and cytoplasm of transfected neonatal rat cardiomyocytes. ANKRD1/CARP is enriched in the insoluble myofibrillar bound fraction (Torrado et al, 2005a;Hayashi et al, 2008), consistent with its strong binding to titin and myopalladin. Although additional studies are required to determine the significance of ANKRD1/CARP subcellular localization, it has been proposed Miller et al, 2004) that ANKRD1/CARP can function: (1) as a component of a titinassociated stretch-sensing complex in the myofibril and (2) as a co-factor of YB-1-regulated transcription in the nucleus.…”

Is titin a ‘winding filament’? A new twist on muscle contraction

“…920 nm) includes the C-terminal and A-band FNIII and Ig domains, PEVK and part of the N2A region [37]. The N2A region contains an epitope that binds p94/calpain3 [38]. It is thought that the T2 fragment forms when p94/calpain3 digests titin near this binding site in the N2A region.…”