The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

Abstract

Obscurin is a large (ϳ800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo. INTRODUCTIONThe three largest proteins of the sarcomere, titin, nebulin, and obscurin, are critical for the assembly of myofibrils and the structural integrity of the sarcomere. The organization of actin and myosin in the mature myofibril is regulated by nebulin and titin, respectively, but the functions of obscurin, the most recently discovered of the three, are still poorly understood. An orthologue of Unc89 (Benian et al., 1996;Sutter et al., 2004), obscurin was first identified in mammals as a ligand of titin. Like titin, it is a multidomain protein (Young et al., 2001) composed largely of immunoglobulinlike (Ig) domains, which, with two fibronectin-like III (FnIII) domains, comprise its N-terminal and central regions. In the A isoform of obscurin, the C-terminal region of obscurin contains a calmodulin-binding (IQ) motif, followed by several Ig domains, a Src homology (SH) 3 domain, a rhoguanine nucleotide exchange factor (rhoGEF) domain, and tandem pleckstrin homology (PH) domain, two more Ig domains, and some nonmodular sequence. The nonmodular region at the extreme carboxy terminus includes several putative phosphorylation sites for extracellular signal-regulated kinase (ERK) kinases, as well as a binding site for a small, integral membrane form of ankyrin that concentrates in the network sarcoplasmic reticulum (nSR) (Young et al., 2001;Russell et al., 2002;Bagnato et al., 2003; KontrogianniKonstantopoulos et al., 2003;Borzok et al., 2007). This ...

Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

“…Moreover, it has been previously shown that ectopic expression of the obscurin RhoGEF motif in COS-7 cells or mouse tibialis anterior muscle results in increased GTP-bound RhoA [36]. Therefore, we hypothesized that in MCF10A breast epithelial cells, knockdown of giant obscurins would result in decreased RhoA activity and a concomitant reduction of RhoA-driven processes downstream of the Rho Activated Kinase (ROCK).…”

Cancer-Related PRUNE2 Protein Is Associated with Nucleotides and Is Highly Expressed in Mature Nerve Tissues

“…The BCH domain in the C-terminal region of PRUNE2 has been reported to inhibit RhoA and cellular transformation mediated by Lbc RhoGEF (Soh and Low 2008). On the other hand, OBSCN protein was reported to possess RhoGEF activity and to activate RhoA (Bowman et al 2008;Ford-Speelman et al 2009). It is possible that PRUNE2 and OBSCN may competitively regulate RhoA activity, thus contributing to the characteristics of LMS and GIST cells.…”

Obscure functions: the location–function relationship of obscurins

“…The RhoGEF domain of obscurin interacts and colocalizes with RhoA, a small GTPase, at the M-band (FordSpeelman et al 2009). Exogenous expression of the obscurin RhoGEF domain in adult skeletal muscle activates RhoA, resulting in increased GTP-bound RhoA and a partial redistribution of RhoA to the cytoplasm, the I-band, and the Z-disc (Ford-Speelman et al 2009). A downstream target of RhoA, Rho associated coiled-coil containing kinase 1 (ROCK1), which functions in a variety of cellular activities, including actin cytoskeletal organization, cell adhesion, and proliferation, also exhibited increased activation in the same system (FordSpeelman et al 2009).…”

“…In addition, giant obscurins expressing the RhoGEF epitope are significantly increased in murine models of aortic constriction (Borisov et al 2003), and increased RhoA and ROCK1 activity have been observed in murine models of cardiac hypertrophy (Ford-Speelman et al 2009). Taken together, these findings support a role of the obscurin RhoGEF domain in regulating RhoA activity and suggest possible signaling functions for obscurins in the response to myocyte injury.…”