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Abstract
We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.
Highlights
The LIM domain is a recently identified cysteine-rich motif defined by 50 – 60 amino acids with the consensus sequence CX2CX16 –23HX2CX2CX2CX16 –21CX2(C/H/D), which contains two closely associated zinc-binding modules [1,2,3]
We report the cloning and characterization of a novel striated muscle-restricted LIM domain-containing protein that we have named Cypher owing to its homology to another LIM domain protein, Enigma
Cloning cypher—To clone novel LIM domain-containing gene(s) that might be important for cardiac development and/or function, the amino acid sequence of the LIM-only protein MLP [13] was used to search the mouse EST data base via the BLAST search program [14]
Summary
The LIM domain is a recently identified cysteine-rich motif defined by 50 – 60 amino acids with the consensus sequence CX2CX16 –23HX2CX2CX2CX16 –21CX2(C/H/D), which contains two closely associated zinc-binding modules [1,2,3]. One group, designated as homeodomain-containing LIM proteins, which includes founder family members LIN-11, ISL1, and MEC-3, has both LIM domain(s) and a homeodomain. Some LIM domains are nuclear proteins involved in cell lineage determination and pattern formation during development [8]. Genetic studies have demonstrated important roles of LIM domain proteins in cell lineage determination, cytoskeletal structure, and organogenesis. It has been shown that mice lacking muscle LIM protein (MLP/CRP3) develop dilated cardiomyopathy with hypertrophy and heart failure after birth [12]. We hypothesized that other LIM domain-containing proteins may play important roles in cardiac function. Characterization of these proteins will help us to better understand the function of LIM domains and may identify candidate genes involved in cardiomyopathies. We report the cloning and characterization of a novel striated muscle-restricted LIM domain-containing protein that we have named Cypher owing to its homology to another LIM domain protein, Enigma
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