The rho-guanine nucleotide exchange factor domain of obscurin activates rhoA signaling in skeletal muscle

Abstract

Obscurin is a large ( approximately 800-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.

Figure 1.

The rhoGEF domain of obscurin interacts with the catalytically inactive form of rhoA and activates wt-rhoA in vitro. (A) COS-7 cells were cotransfected with plasmids encoding GFP-tagged rhoGEF-PH domains of obscurin (left, shown in green on the right) and HA-tagged, catalytically inactive forms of rhoA, cdc42 or rac1 (rhoA-DN, cdc42-DN or rac1-DN; center, shown in red on the right). HA-rhoA-DN colabels extensively with GFP-rhoGEF-PH, but HA-cdc42-DN and rac1-DN do not (right, shown in yellow, insets). Bars, 10 μm. (B) COS-7 cells were cotransfected as in A, or with plasmids encoding GFP and HA-rhoA-DN or GFP-rhoGEF-PH and HA-rhoA-CA. After transfection, rabbit anti-GFP antibody was used to immunoprecipitate GFP or GFP-rhoGEF-PH from cell lysates. Western blot analysis reveals that HA-rhoA-DN coimmunoprecipitates with the GFP-rhoGEF-PH domains of obscurin but that HA-rhoA-CA and HA-cdc42-DN do not. (C) COS-7 cells were cotransfected with plasmids encoding wt-rhoA and GFP, GFP-rhoGEF, or GFP-rhoGEF-PH. After transfection, GST-tagged rhotekin binding domain (GST-RBD) was used to adsorb active, GTP-bound rhoA from cell lysates. Western blot analysis shows that coexpression of GFP-rhoGEF or GFP-rhoGEF-PH increases the amount of active rhoA, compared with GFP alone.

Figure 2.

RhoA is expressed and colocalizes with the rhoGEF domain of obscurin in primary myotubes differentiating in culture. (A) Total protein from homogenates of primary myotubes was analyzed as a function of time in culture by Western blotting for rhoA and obscurin. RhoA expression increases up to day 5 in culture (top), at which time the large, ∼800-kDa isoform of obscurin is clearly expressed (middle). Smaller bands that label with antibody to the rhoGEF domain of obscurin are expressed at apparently constant levels between D2 and D5 in culture (bottom). (B) Primary myotube cultures were labeled at 4, 6, or 7 d after initial plating with primary antibodies for the Z-disk region of titin (titinZ, green), rhoA (red), and the rhoGEF domain of obscurin (blue). RhoA organizes with obscurin at the M-band after the ∼800-kDa form of obscurin is expressed (overlay, purple). Bars, 10 μm.

Figure 3.

RhoA colocalizes with the rhoGEF domain of obscurin at the M-band of adult skeletal muscle. (A) Enzymatically dissociated flexor digitorum brevis muscle fibers from a young adult rat were labeled for the carboxy terminus of titin (titinM) or the amino terminus of titin (titinZ; green), rhoA or the amino terminus of obscurin (ObsNt; red), and the rhoGEF domain of obscurin (blue). (B) Longitudinal cryosections (20 μm) of mouse TA muscle were labeled with primary antibodies to rhoA (shown in red) and the carboxy terminus of titin (titinM) or the amino terminus of titin (titinZ), shown in green. (C) Longitudinal cryosections (20 μm) of mouse TA muscle were labeled with primary antibodies to rhoA, cdc42, or rac1 (red), and the rhoGEF domain of obscurin (green). RhoA codistributes with obscurin's rhoGEF domain at the M-band in both preparations (shown in purple in A; yellow in B and C). Cdc42 is present predominantly at the Z-disk, and rac1 at the A-band. Bars, 10 μm. (D) COS-7 cells were cotransfected with HA-tagged, wild-type forms of rhoA, cdc42, or rac1. The HA-tagged proteins were extracted and immunoprecipitated with antibody to the HA tag. Western blot analysis shows that the rhoA antibody used in this study is specific for rhoA and does not recognize two closely related proteins, cdc42 or rac1 and that antibodies to the latter proteins do not recognize rhoA. (E) COS-7 cells were cotransfected with HA-tagged forms of rhoA or tc10. The HA tagged proteins were extracted, immunoprecipitated and analyzed by Western blot as described in D. The rhoA antibody used in these experiments does not recognize tc10.

Figure 4.

Overexpression of obscurin's rhoGEF domain alters rhoA localization in vivo. (A) Cross sections (20 μm) from 7-wk-old rat TA muscles were examined 1 wk after IVGT of GFP-rhoGEF or GFP-rhoGEF-PH, induced by electroporation. IVGT resulted in ∼60% of the fibers expressing GFP-rhoGEF, ∼30% expressing GFP-rhoGEF-PH, and ∼80% expressing GFP. (B) Longitudinal cryosections (20 μm) from rat TA were examined as described in A. The contralateral TA, transfected via IVGT with GFP alone, was used as a control. Top, after transfection of a vector encoding GFP, endogenous rhoA remains localized to the M-band (red; purple in merged image to right). Obscurin's rhoGEF localizes to both the M-band and the Z-disk or Z-I junction (blue; purple in merged image to right). Middle, transfection of GFP-rhoGEF results in rhoA localization at both the M-band and Z-disks or Z-I junctions (red). Bottom, GFP-rhoGEF-PH expression promotes more extensive association of rhoA with Z-disks and Z/I junction (red). The rhoGEF antibody recognizes both endogenous obscurin and the GFP-rhoGEF fusion proteins. Bars, 10 μm. (C) Quantification of rhoA intensity, measured with ImageJ software across one sarcomere length from the insets shown in B. Top, intensity of rhoA staining in GFP transfected muscle, from Z-disk to M-band to Z-disk. Middle, intensity of rhoA staining in GFP-rhoGEF transfected muscle. Bottom, intensity of rhoA staining in GFP-rhoGEF-PH transfected muscle. RhoA localizes to both the M-band and the Z-disk in the presence of GFP-rhoGEF or GFP-rhoGEF-PH.

Figure 5.

Overexpression of obscurin's rhoGEF domain increases rhoA expression and activity in vivo and alters expression of two downstream effectors of rhoA, ROCK1 and CRIK. (A) Homogenates from rat TA transfected by IVGT with plasmids encoding GFP or GFP-rhoGEF were analyzed for rhoA activity and expression. Top, RhoA activity was assayed with a GST fusion protein containing the rhotekin binding domain (GST-RBD), which binds specifically to the GTP-bound form of rhoA, followed by Western blotting. Middle, Western blotting was used to determine the relative levels of rhoA in the homogenates used for the GST-RBD pull-down assays. Bottom, labeling by Ponceau Red indicates equal protein loading in the two samples. (B) Quantification of the results shown in A (and data not shown), expressed as the ratio of samples transfected with GFP-rhoGEF compared with those transfected with GFP alone, shows an approximately threefold increase in rhoA levels and an ∼12-fold increase in active rhoA when the rhoGEF domain is overexpressed (n = 3). These differences were significant (**p < 0.005). (C) After IVGT, as described in A, homogenates of transfected muscles were analyzed by Western blotting for expression of ROCK1 and CRIK. Overexpression of the rhoGEF domain of obscurin results in increased levels of ROCK1 and decreased levels of CRIK (top). The levels of small GTPases rac1 and cdc42 do not change in samples that overexpress the rhoGEF domain of obscurin (bottom). As described above, Ponceau Red staining verifies equal loading of the two samples. (D) Quantification of the differences illustrated in C, determined by comparison of samples expressing GFP and GFP-rhoGEF, shows a 3.6-fold increase in ROCK1 and a 2.4-fold decrease in CRIK expression when GFP-rhoGEF is overexpressed in adult rat TA. The differences for both ROCK1 and CRIK are statistically significant (*p < 0.05 and **p < 0.005, respectively).

Figure 6.

Obscurin's rhoGEF domain alters localization of ROCK1 and CRIK in vivo. Longitudinal cryosections (20 μm) from rat TA were examined as described in Figure 4B. The contralateral TA, transfected via IVGT with plasmid encoding GFP, was used as a control. (A) Top, after transfection of a vector encoding GFP, ROCK1 primarily localizes to the Z-disk (blue). RhoA localizes to the M-band (red). Bottom, transfection of GFP-rhoGEF results in increased labeling for ROCK1, and its localization to the Z-disk and Z/I junction, with some staining at the M-band. RhoA localizes to both the M-band and the I-band, and possibly the Z-disk (red). (B) After transfection of a vector encoding GFP, CRIK localizes to the M-band and A-band regions (blue). Bars, 10 μm.

Figure 7.

Contraction-induced injury alters rhoA localization and activates rhoA in vivo. Tissue from injured or contralateral uninjured rat hindlimbs was collected immediately after large-strain lengthening contractions. (A) Longitudinal cryosections (20 μm) of rat TA were analyzed by confocal fluorescence microscopy. Top, control. Absence of FDX indicates lack of injury to the muscle fiber. RhoA localizes to the M-band (red), and obscurin's rhoGEF domain localizes to the M-band and the Z-disk (blue). Bottom, injured: FDX-positive fibers indicate that they were injured (green). RhoA localizes to the M-band, Z-disk and Z/I junction (red), whereas obscurin localizes to the M-band and the Z-disk (blue). (B) Quantification of rhoA intensity, measured with ImageJ software across one sarcomere length, from the insets shown in A. Top, rhoA in uninjured muscle, from Z-disk to M-band to Z-disk. Bottom, rhoA in injured muscle. (C) Homogenates from control (uninjured) or injured (D0) rat TA were analyzed for rhoA expression and activity. Top, Western blotting was used to determine the relative levels of rhoA in the homogenates. Bottom, RhoA activity in the homogenates shown in the top panel was assayed with a GST fusion protein containing the RBD (GST-RBD), which binds specifically to the GTP-bound form of rhoA, followed by Western blotting. Total active rhoA is greater in injured tissue than in control tissue, but the total amount of rhoA remains unchanged immediately after injury.