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. 2008 Mar 28;283(13):8070-4.
doi: 10.1074/jbc.C700221200. Epub 2008 Feb 5.

Deregulated Protein Kinase A Signaling and Myospryn Expression in Muscular Dystrophy

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Free PMC article

Deregulated Protein Kinase A Signaling and Myospryn Expression in Muscular Dystrophy

Joseph G Reynolds et al. J Biol Chem. .
Free PMC article

Abstract

Alterations in signaling pathway activity have been implicated in the pathogenesis of Duchenne muscular dystrophy, a degenerative muscle disease caused by a deficiency in the costameric protein dystrophin. Accordingly, the notion of the dystrophin-glycoprotein complex, and by extension the costamere, as harboring signaling components has received increased attention in recent years. The localization of most, if not all, signaling enzymes to this subcellular region relies on interactions with scaffolding proteins directly or indirectly associated with the dystrophin-glycoprotein complex. One of these scaffolds is myospryn, a large, muscle-specific protein kinase A (PKA) anchoring protein or AKAP. Previous studies have demonstrated a dysregulation of myospryn expression in human Duchenne muscular dystrophy, suggesting a connection to the pathophysiology of the disorder. Here we report that dystrophic muscle exhibits reduced PKA activity resulting, in part, from severely mislocalized myospryn and the type II regulatory subunit (RIIalpha) of PKA. Furthermore, we show that myospryn and dystrophin coimmunoprecipitate in native muscle extracts and directly interact in vitro. Our findings reveal for the first time abnormalities in the PKA signal transduction pathway and myospryn regulation in dystrophin deficiency.

Figures

FIGURE 1.
FIGURE 1.
Impaired myospryn protein expression and localization in mdx mice. A, left panel, Western blot analysis of myospryn protein in wild type (WT) and mdx hind limb muscle. WT (lanes 1–5) and mdx (lanes 6–9) samples were immunoblotted (IB) with anti-myospryn. (*, degradation products.) Right panel, quantification of full-length myospryn. Signal intensity was quantified by ImageQuant (GE Healthcare), and the background was subtracted for each sample. Signal intensities for each group were averaged and normalized to WT set at 100 ± S.D. WT = 100 ± 48%, mdx = 27 ± 23%. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, double label immunohistochemistry on wild type and mdx mouse hind limb muscle (transverse sections). Left panels, anti-vinculin antibody delineates the myofiber periphery. Note that the costameric protein vinculin is largely unaffected in mdx hind limb muscle. Middle panels, anti-myospryn antibody reveals severe mislocalization of myospryn in mdx tissue. Right panels, merged images for vinculin and myospryn. (*, myofiber cytoplasm; ↑, myofiber periphery/sarcolemma; >, punctuate foci of myospryn staining in mdx muscle.)
FIGURE 2.
FIGURE 2.
Association between dystrophin and myospryn. A, dystrophinmyospryn complex in native hind limb muscle. Protein extracts were immunoprecipitated with anti-myospryn or anti-GAL4 antibodies and immunoblotted (IB) using anti-dystrophin antibodies (427 kDa, endogenous dystrophin). B, myospryn and dystrophin colocalize at the muscle costamere. Double immunohistochemistry of transverse sections from mouse hind limb muscle is shown. Upper panel, dystrophin localizes to myofiber periphery. Middle panel, myospryn localizes to peripheral myofiber as described previously (28). Lower panel, merged images indicating co-localization. C, direct interaction between myospryn and dystrophin. Western blot analysis of FLAG-dystrophin (Dp71) protein extracts subjected to a GST pulldown assay with myospryn (GST-Spe) or GST alone is shown. D, mapping the dystrophin interaction regions in myospryn. Left panel, Western analysis of coIP assays with several Myc-myospryn constructs co-transfected with FLAG-dystrophin (Dp71). Right panel, schematic of myospryn depicting dystrophin interaction regions.
FIGURE 3.
FIGURE 3.
Deregulated PKA signaling and regulation of myospryn gene expression by PKA-CREB. A, double label immunohistochemistry of wild type and mdx hind limb muscle (transverse sections). Left panels, anti-vinculin immunoreactivity along myofiber periphery. Middle panels, anti-RII reveals mislocalization of this PKA regulatory subunit in mdx muscle. Right panels, merged images for vinculin and RII immunostaining. (*; myofiber cytoplasm. ^; sarcolemma. >; punctuate foci in mdx muscle). B, reduced PKA activity in mdx hind limb muscle. Protein extracts from wild type (n = 3) and mdx (n = 3) muscle were subjected to a PKA activity assay. The difference in group means was significant (p < 0.003). C, PKA subunit expression in wild type and mdx muscle. Immunoblots of protein extracts from wild type (n = 4) and mdx (n = 4) hind limb muscle demonstrating the predominant catalytic (cat) and regulatory PKA subunits are shown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, quantitative RT-PCR of AKAP expression. cDNA reactions from pooled mdx hind limb muscle mRNA (n = 3) relative to wild type were normalized to 1.0. All samples were performed in triplicate, and Ct values for each gene were averaged and normalized to glyceraldehyde-3-phosphate dehydrogenase. E, myospryn is transcriptionally activated by PKA-CREB signaling. Transient transfection reporter assays in COS cells of the myospryn reporter containing the wild type CRE (TAACGTTA) and a myospryn reporter with a mutant (mut) CRE (TCCCGGGA) were performed. Luciferase data were performed in triplicate, and the difference in promoter activity was significant (p < 0.01).

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