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Suzuki, Suguru; Ohgaki, Masako; Ichiyanagi, Masayuki; Ozawa, Masakuni

doi:10.1023/a:1006526915828

Cited by 38 publications

(12 citation statements)

References 10 publications

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“…β 1 ‐Syntrophin, a dystrophin associated protein, was also demonstrated by the same assay to interact with the C‐terminal dystrophin fragment (data not shown). This result was consistent with findings by our group [16] and others using different methods [22].…”

Section: Resultssupporting

confidence: 94%

In vitro expressed dystrophin fragments do not associate with each other

Chan

Kunkel

1997

FEBS Letters

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Dystrophin, a component of the muscle membrane cytoskeleton, is the protein altered in Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Dystrophin shares significant homology with other cytoskeletal proteins, such as oc-actinin and spectrin. On the basis of its sequence similarity with a-actinin and spectrin, dystrophin has been proposed to function as dimer. However, the existence of both dimers and monomers have been observed by electron microscopy. To address this apparent discrepancy, we expressed dystrophin fragments composed of different domains in an in vitro translation system. The expressed fragments were tested for their ability to interact with each other and full-length dystrophin by both immunoprecipitation and blot overlay assays. These assays were successfully used to demonstrate the dimerization of a-actinin and spectrin, yet failed to detect any interaction between dystrophin fragments. Although these in vitro results do not prove that dystrophin is not a dimer in vivo, they do indicate that this interaction is not like that of the a-actinin and spectrin.

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Section: Resultssupporting

confidence: 94%

In vitro expressed dystrophin fragments do not associate with each other

Chan

Kunkel

1997

FEBS Letters

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“…In agreement with expression experiments of fusion proteins containing different C‐terminal fragments of dystrophin [7,8,20,21] we observed more than one GST fusion protein in each expression experiment indicating degradation during expression/purification (Fig. 2).…”

Section: Resultssupporting

confidence: 86%

“…Constructs including either WW, EF hands or ZZ domains were very susceptible to endogenous proteases especially in the linker following the GST protein. This protease susceptibility was already described for the corresponding dystrophin fusion constructs [7,8,20,21]. GST fusion proteins were induced with 1 mM isopropyl‐1‐thio‐β‐ D ‐galactopyranoside (IPTG, Sigma‐Aldrich) for only 2 h at 37°C to reduce protein degradation.…”

Section: Methodsmentioning

confidence: 99%

Contribution of the different modules in the utrophin carboxy‐terminal region to the formation and regulation of the DAP complex

et al. 2000

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The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein^protein interaction. These domains (WW, EF hands, ZZ and H1^H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxyterminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in L L-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to L Ldystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin L L-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca 2 -dependent binding of calmodulin. The remaining two motifs found in the carboxyterminus (H1^H2) mediate the formation of utrophin^dystro-brevin hybrids but do not select ligands in a repertoire of random nonapeptides.z 2000 Federation of European Biochemical Societies.

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“…The region of dystrobrevin which binds to syntrophin has been mapped within the CRCT (Fig. 1) [38–40]. To determine whether the PYCT domain altered the association of dystrobrevin with syntrophin, syntrophin was immunoprecipitated from rat brain homogenate using a monoclonal antibody which recognizes all three isoforms of syntrophin.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the tyrosine phosphorylation and distribution of dystrobrevin isoforms

1998

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Dystrobrevin, a member of the dystrophin family of proteins, was initially identified as a major tyrosine phosphorylated synaptic protein in the electric organ of Torpedo californica. In this paper, we show that the major sites of tyrosine phosphorylation of Torpedo dystrobrevin are within its Cterminus, on Tyr-693 and Tyr-710. Cloning of the mammalian homologue of dystrobrevin has recently shown that this phosphotyrosine containing tail, or PYCT, is subject to alternative splicing. To compare the expression and distribution of PYCT 3 and PYCT + splice variants, we generated antibodies against different regions of dystrobrevin. Here we show that the PYCT 3 isoform of 62 kDa is expressed at high levels in all tissues examined. In contrast, PYCT + isoforms are expressed primarily in brain and muscle, where they are concentrated at synapses. Moreover, PYCT + isoforms associate more tightly with the membrane and with syntrophin, another synaptically enriched protein. These results suggest that PYCT + isoforms of dystrobrevin are specialized components of the dystroglycan complex which render the complex sensitive to regulation by tyrosine kinases.z 1998 Federation of European Biochemical Societies.

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Untitled

Cited by 38 publications

References 10 publications

In vitro expressed dystrophin fragments do not associate with each other

In vitro expressed dystrophin fragments do not associate with each other

Contribution of the different modules in the utrophin carboxy‐terminal region to the formation and regulation of the DAP complex

Characterization of the tyrosine phosphorylation and distribution of dystrobrevin isoforms

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