Identification of calmodulin-binding proteins in chicken embryo fibroblasts.

Burgess, Wilson H.; Watterson, Daniel; Eldik, Linda J. Van

doi:10.1083/jcb.99.2.550

Cited by 64 publications

(20 citation statements)

References 46 publications

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“…In agreement with this relationship between structure and function, KRP does not have protein kinase activity with myosin light chain as a substrate and does not have calmodulin-binding activity as determined by calmodulin-Sepharose chromatography (63) or gel overlay analysis (11). As expected on the basis of the segmental-organization model of MLCK described by Shoemaker et al (50), KRP (up to 4 ,uM) did not have significant effects on MLCK-catalyzed phosphotransferase activity with the myosin light-chain substrate.…”

Section: Methodsmentioning

confidence: 74%

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

Collinge¹,

Matrisian²,

Zimmer³

et al. 1992

Mol. Cell. Biol.

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We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.

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Section: Methodsmentioning

confidence: 74%

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

Collinge¹,

Matrisian²,

Zimmer³

et al. 1992

Mol. Cell. Biol.

Self Cite

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“…258 Following SDS/polyacrylamide-gel electrophoresis, proteins were renatured, and an '25I-labelled calmodulin overlay was performed as described [166]. The arrow indicates the subunits of CaM-kinase II (51/53 kDa) [15,16].…”

Section: Species Tissue and Subcellular Distributionmentioning

confidence: 99%

Calcium/Calmodulin-Dependent Protein Kinase II

Colbran

Soderling

1990

Current Topics in Cellular Regulation

174

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“…[Burgess, et al, 1984;Carlin, Grab, and Siekevitz, 1983;Nagle and Burnside, 19841. The average M, of the highmolecular-weight calmodulin-binding polypeptide of coelomocytes was 240,000, but estimated values ranged from 226,000 to 252,000.…”

Section: Discussionmentioning

confidence: 98%

Calmodulin and caimodulin‐binding proteins in the morphological transformation of sea urchin coelomocytes

Venuti

Edds

1986

Cell Motil. Cytoskeleton

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Sea urchin coelomocytes contain an actin-based cytoskeleton that undergoes major organizational changes as the cells transform from one morphology (petaloid) to another (filopodial). The molecular mechanisms directing and regulating this cytoskeletal reorganization are not well understood; Ca2+ has been implicated, but the specific targets of its action have not been identified. Since the effect of Ca2+ on a variety of cellular processes has been shown to be mediated by the Ca2+-binding protein calmodulin, we investigated the role of this protein in coelomocyte morphological transformation. The calmodulin inhibitory drugs trifluoperazine, chlorpromazine, and calmidazolium were shown to inhibit coelomocyte morphological transformation in response to hypotonic shock in a dosedependent fashion and at concentrations consistent with their reported potencies as anti-calmodulin agents. Calmodulin isolated from coelomocytes using trifluorophenothiazine affinity chromatography co-migrates with bovine brain calmodulin on 15% SDS-polyacrylamide gels and demonstrates a characteristic shift in electrophoretic mobility in the presence of Ca2+. Another diagnostic for calmodulin, Ca2+-dependent activation of exogenous 3'15' cyclic AMP phosphodiesterase, was demonstrated by whole coelomocyte homogenates, heat-treated homogenates, and the affinity purified coelomocyte protein. Localization of calmodulin in coelomocytes by indirect immunofluorescence reveals an association of calmodulin, at least in part, with the actin-based cytoskeleton. Calmodulinbinding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using '251-calmodulin overlay procedures. Ca2+-dependent calmodulin-binding in these preparations was demonstrated for all but the M, 30,000 and 20,000 coelomocyte polypeptides. The majority of the calmodulin-binding proteins identified in whole petaloid coelomocyte preparations are also found in Triton X-100 insoluble cytoskeletal fractions. Immunoblotting with antiserum raised against chicken erythrocyte alpha-spectrin suggests that the 240,000 M, calmodulin-binding polypeptide corresponds to coelomocyte alpha-spectrin. This protein was enriched in isolated coelomocyte filopodia where, we propose, it serves an analogous function to its counterpart in erythrocytes, in linking the actin-cytoskeleton to the plasma membrane. Thus, calmodulin is present in coelomocytes and possibly participates in the morphological transformation of these cells through regulation of cytoskeletal and/or membrane-cytoskeletal interactions.

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Identification of calmodulin-binding proteins in chicken embryo fibroblasts.

Cited by 64 publications

References 46 publications

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

Calcium/Calmodulin-Dependent Protein Kinase II

Calmodulin and caimodulin‐binding proteins in the morphological transformation of sea urchin coelomocytes

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