In vitro expressed dystrophin fragments do not associate with each other

Chan, Yiu-mo; Kunkel, Louis M.

doi:10.1016/s0014-5793(97)00454-7

Cited by 23 publications

(20 citation statements)

References 34 publications

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“…In previous studies with blot overlay and coprecipitation experiments, we had detected no evidence of dystrophin homodimerization (18). To be sure, we tested the possibility of homodimerization of both dystrophin and dystrobrevin through their coiled-coil motif in the two-hybrid system.…”

Section: Discussionmentioning

confidence: 96%

Dystrobrevin and dystrophin: An interaction through coiled-coil motifs

Puccio

Rajala

Kunkel

1997

Proc. Natl. Acad. Sci. U.S.A.

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174

161

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Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins' homologous coiled-coil motifs that f lank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiledcoil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.

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Section: Discussionmentioning

confidence: 96%

Dystrobrevin and dystrophin: An interaction through coiled-coil motifs

Puccio

Rajala

Kunkel

1997

Proc. Natl. Acad. Sci. U.S.A.

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174

161

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“…The blots were probed with 35 Slabeled hDlg or GST-PDZ1-2, washed extensively, and dried for autoradiography as described (22).…”

Section: Methodsmentioning

confidence: 99%

Characterization of PDZ-binding kinase, a mitotic kinase

Gaudet

Branton²,

Lue³

2000

Proc. Natl. Acad. Sci. U.S.A.

195

218

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hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine͞threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T͞SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T͞SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2͞cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation.

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“…The proteins ␣-actinin-2 or -3 and myozenin were produced by IVTT of the complete coding sequences cloned into the expression vectors pMGT1 and pFHR3 (21) by using the TNT T7-coupled reticulocyte lysate system (Promega) in 50-l reactions per the manufacturer's protocol. The C-terminal region (residues 2,191-2,705) of ␥-filamin was expressed from clone 2-14, and partial dystrophin fragments were as described (5,22). pFHR3 introduces an N-terminal FLAG nonapeptide (MDYKDDDDK) epitope-tag that was recognized by using M2 Abs (Eastman Kodak).…”

Section: Methodsmentioning

confidence: 99%

Myozenin: An alpha -actinin- and gamma -filamin-binding protein of skeletal muscle Z lines

Takada¹

2001

Proceedings of the National Academy of Sciences

121

123

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To better understand the structure and function of Z lines, we used sarcomeric isoforms of ␣-actinin and ␥-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an ␣-actinin-and ␥-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by ␣-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for ␣-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.

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In vitro expressed dystrophin fragments do not associate with each other

Cited by 23 publications

References 34 publications

Dystrobrevin and dystrophin: An interaction through coiled-coil motifs

Dystrobrevin and dystrophin: An interaction through coiled-coil motifs

Characterization of PDZ-binding kinase, a mitotic kinase

Myozenin: An alpha -actinin- and gamma -filamin-binding protein of skeletal muscle Z lines

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