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Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.

Zhou Q, Chu PH, Huang C, Cheng CF, Martone ME, Knoll G, Shelton GD, Evans S, Chen J - J. Cell Biol. (2001)

Bottom Line: Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function.In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components.These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Medicine, University of California at San Diego School of Medicine, La Jolla, CA 92093, USA.

ABSTRACT
Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH(2) terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

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Interaction of the PDZ domain of Cypher requires the COOH terminus of α-actinin. (A) Schematic diagram of α-actinin 2. The actin binding domain (ABD, aa 1–273), spectrin-like repeats (SLR, aa 274–755), and EF hands (aa 755–894) are indicated. B. Autoradiogram of 35S methionine-labeled proteins after coprecipitated with GST-PDZ glutathione resin (see Materials and Methods). Lane 1, full-length α-actinin 2; lane 2, Spectrin repeats; lane 3, ABD domain; lane 4, the four EF hands. Exposure time for the autoradiogram was 4 h at room temperature. The bottom panel shows the input of each in vitro translated protein. Exposure time for the autoradiogram was 20 min at room temperature. Strongest binding to the PDZ domain was observed with the COOH-terminal four EF hands of α-actinin 2 (lane 4). C. Autoradiogram of 35S methionine-labeled α-actinin 2 COOH-terminal fragments after coincubation and precipitation with GST-PDZ fusion proteins. Lane 1, 35S- labeled four EF hands (4EF, aa 756 to 894); lane 2, four EF hands deleted for the three COOH-terminal amino acids (4EF-del3, aa 756 to 891); lane 3, first two EF hands (EF1-2, aa 756–821); lane 4, last two EF hands (EF3-4, aa 822–893). Exposure time for the autoradiogram was 3 h at room temperature. The bottom panel shows 20% input of in vitro–translated protein. Exposure time for the autoradiogram was 30 min at room temperature. Deletion of the last two EF hands, including the three terminal amino acids, or deletion of the three terminal amino acids alone, abolished binding to the PDZ domain (lanes 2 and 3). (D) Autoradiogram of [35S]methionine-labeled proteins after coprecipitation with GST-PDZ glutathione resin. Lane 1, full-length α-actinin 2; lane 2, α-actinin 2 deleted for the three COOH-terminal amino acids (actinin-del3); lane 3 and 4, 20% input of in vitro–translated full-length α-actinin 2 and actinin-del3 proteins. Exposure time for the autoradiogram was 3 h at room temperature.
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fig7: Interaction of the PDZ domain of Cypher requires the COOH terminus of α-actinin. (A) Schematic diagram of α-actinin 2. The actin binding domain (ABD, aa 1–273), spectrin-like repeats (SLR, aa 274–755), and EF hands (aa 755–894) are indicated. B. Autoradiogram of 35S methionine-labeled proteins after coprecipitated with GST-PDZ glutathione resin (see Materials and Methods). Lane 1, full-length α-actinin 2; lane 2, Spectrin repeats; lane 3, ABD domain; lane 4, the four EF hands. Exposure time for the autoradiogram was 4 h at room temperature. The bottom panel shows the input of each in vitro translated protein. Exposure time for the autoradiogram was 20 min at room temperature. Strongest binding to the PDZ domain was observed with the COOH-terminal four EF hands of α-actinin 2 (lane 4). C. Autoradiogram of 35S methionine-labeled α-actinin 2 COOH-terminal fragments after coincubation and precipitation with GST-PDZ fusion proteins. Lane 1, 35S- labeled four EF hands (4EF, aa 756 to 894); lane 2, four EF hands deleted for the three COOH-terminal amino acids (4EF-del3, aa 756 to 891); lane 3, first two EF hands (EF1-2, aa 756–821); lane 4, last two EF hands (EF3-4, aa 822–893). Exposure time for the autoradiogram was 3 h at room temperature. The bottom panel shows 20% input of in vitro–translated protein. Exposure time for the autoradiogram was 30 min at room temperature. Deletion of the last two EF hands, including the three terminal amino acids, or deletion of the three terminal amino acids alone, abolished binding to the PDZ domain (lanes 2 and 3). (D) Autoradiogram of [35S]methionine-labeled proteins after coprecipitation with GST-PDZ glutathione resin. Lane 1, full-length α-actinin 2; lane 2, α-actinin 2 deleted for the three COOH-terminal amino acids (actinin-del3); lane 3 and 4, 20% input of in vitro–translated full-length α-actinin 2 and actinin-del3 proteins. Exposure time for the autoradiogram was 3 h at room temperature.

Mentions: To define sites within α-actinin 2 which bind to the PDZ domain, we made a series of α-actinin 2 deletion constructs which were translated in vitro in the presence of [S35]-methionine and subsequently assayed for binding to GST–PDZ fusion proteins. Results of these assays demonstrated that the COOH terminus of α-actinin 2, containing four degenerate EF hands (aa 756–894), bound strongly to the Cypher PDZ domain (Fig. 7 B).


Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.

Zhou Q, Chu PH, Huang C, Cheng CF, Martone ME, Knoll G, Shelton GD, Evans S, Chen J - J. Cell Biol. (2001)

Interaction of the PDZ domain of Cypher requires the COOH terminus of α-actinin. (A) Schematic diagram of α-actinin 2. The actin binding domain (ABD, aa 1–273), spectrin-like repeats (SLR, aa 274–755), and EF hands (aa 755–894) are indicated. B. Autoradiogram of 35S methionine-labeled proteins after coprecipitated with GST-PDZ glutathione resin (see Materials and Methods). Lane 1, full-length α-actinin 2; lane 2, Spectrin repeats; lane 3, ABD domain; lane 4, the four EF hands. Exposure time for the autoradiogram was 4 h at room temperature. The bottom panel shows the input of each in vitro translated protein. Exposure time for the autoradiogram was 20 min at room temperature. Strongest binding to the PDZ domain was observed with the COOH-terminal four EF hands of α-actinin 2 (lane 4). C. Autoradiogram of 35S methionine-labeled α-actinin 2 COOH-terminal fragments after coincubation and precipitation with GST-PDZ fusion proteins. Lane 1, 35S- labeled four EF hands (4EF, aa 756 to 894); lane 2, four EF hands deleted for the three COOH-terminal amino acids (4EF-del3, aa 756 to 891); lane 3, first two EF hands (EF1-2, aa 756–821); lane 4, last two EF hands (EF3-4, aa 822–893). Exposure time for the autoradiogram was 3 h at room temperature. The bottom panel shows 20% input of in vitro–translated protein. Exposure time for the autoradiogram was 30 min at room temperature. Deletion of the last two EF hands, including the three terminal amino acids, or deletion of the three terminal amino acids alone, abolished binding to the PDZ domain (lanes 2 and 3). (D) Autoradiogram of [35S]methionine-labeled proteins after coprecipitation with GST-PDZ glutathione resin. Lane 1, full-length α-actinin 2; lane 2, α-actinin 2 deleted for the three COOH-terminal amino acids (actinin-del3); lane 3 and 4, 20% input of in vitro–translated full-length α-actinin 2 and actinin-del3 proteins. Exposure time for the autoradiogram was 3 h at room temperature.
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Related In: Results  -  Collection

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fig7: Interaction of the PDZ domain of Cypher requires the COOH terminus of α-actinin. (A) Schematic diagram of α-actinin 2. The actin binding domain (ABD, aa 1–273), spectrin-like repeats (SLR, aa 274–755), and EF hands (aa 755–894) are indicated. B. Autoradiogram of 35S methionine-labeled proteins after coprecipitated with GST-PDZ glutathione resin (see Materials and Methods). Lane 1, full-length α-actinin 2; lane 2, Spectrin repeats; lane 3, ABD domain; lane 4, the four EF hands. Exposure time for the autoradiogram was 4 h at room temperature. The bottom panel shows the input of each in vitro translated protein. Exposure time for the autoradiogram was 20 min at room temperature. Strongest binding to the PDZ domain was observed with the COOH-terminal four EF hands of α-actinin 2 (lane 4). C. Autoradiogram of 35S methionine-labeled α-actinin 2 COOH-terminal fragments after coincubation and precipitation with GST-PDZ fusion proteins. Lane 1, 35S- labeled four EF hands (4EF, aa 756 to 894); lane 2, four EF hands deleted for the three COOH-terminal amino acids (4EF-del3, aa 756 to 891); lane 3, first two EF hands (EF1-2, aa 756–821); lane 4, last two EF hands (EF3-4, aa 822–893). Exposure time for the autoradiogram was 3 h at room temperature. The bottom panel shows 20% input of in vitro–translated protein. Exposure time for the autoradiogram was 30 min at room temperature. Deletion of the last two EF hands, including the three terminal amino acids, or deletion of the three terminal amino acids alone, abolished binding to the PDZ domain (lanes 2 and 3). (D) Autoradiogram of [35S]methionine-labeled proteins after coprecipitation with GST-PDZ glutathione resin. Lane 1, full-length α-actinin 2; lane 2, α-actinin 2 deleted for the three COOH-terminal amino acids (actinin-del3); lane 3 and 4, 20% input of in vitro–translated full-length α-actinin 2 and actinin-del3 proteins. Exposure time for the autoradiogram was 3 h at room temperature.
Mentions: To define sites within α-actinin 2 which bind to the PDZ domain, we made a series of α-actinin 2 deletion constructs which were translated in vitro in the presence of [S35]-methionine and subsequently assayed for binding to GST–PDZ fusion proteins. Results of these assays demonstrated that the COOH terminus of α-actinin 2, containing four degenerate EF hands (aa 756–894), bound strongly to the Cypher PDZ domain (Fig. 7 B).

Bottom Line: Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function.In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components.These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Department of Medicine, University of California at San Diego School of Medicine, La Jolla, CA 92093, USA.

ABSTRACT
Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH(2) terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with alpha-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

Show MeSH
Related in: MedlinePlus