Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate

Burgess, Wilson H.; Jemiolo, David K.; Kretsinger, Robert H.

doi:10.1016/0005-2795(80)90254-8

Cited by 261 publications

(112 citation statements)

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“…There appears to be a striking difference in electrophoretic mobility between the species of phosphocalmodulin yielded upon phosphorylation with the insulin receptor and the EGF receptor. Non-phosphorylated calmodulin exhibits an altered migration on SDSPAGE in the presence of calcium compared to its mobility in the absence of calcium (Burgess et al, 1980). A similar shift is observed with calmodulin phosphorylated by the EGF-receptor tyrosine kinase (San Jod et al, 1992; this work).…”

Section: Discussionsupporting

confidence: 75%

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Benguría

Hernández‐Perera

Martínez‐Pastor

et al. 1994

European Journal of Biochemistry

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An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3-8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 3ZP/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.Calmodulin is an intracellular calcium receptor that mediates multiple essential functions in eukaryotic cells (Means and Dedman, 1980; Klee et al., 1980;Klee and Vanaman, 1982;Manalan and Klee, 1984;Veigl et al., 1984;Strynadka and James, 1989;Bachs et al., 1992), including the regulation of cell proliferation (Veigl et al., 1984). In this context, it has been demonstrated that this ubiquitous regulator can control multiple nuclear processes (Bachs et al., 1992). Nevertheless, the role of calmodulin in cell proliferation does not appear to be exclusively exerted at the level of the nucleus. Recently we have demonstrated that the epidermal growth factor (EGF) receptor can be isolated by calmodulin-affinity chromatography, and that calmodulin inhibits its tyrosine lunase activity (San JosC et al., 1992). Calmodulin, therefore, could potentially play a regulatory role in the EGF-mediated mitogenic-signal pathway.Calmodulin has been shown to be a substrate for several different protein kinases (Plancke and Lazarides, 1983 ;Hiiring et al., 1985;Fukami et al., 1985;Graves et al., 1986;Lin et al., 1986;Nakajo et al., 1986Nakajo et al., , 1988Meggio et al., 1987Meggio et al., , 1992Kubo and Strott, 1988;Sacks and McDonald, 1988;Laurino et al., 1988; Sacks et al., 1989aSacks et al., , 1992aSan JosC et al., 1992;Benguria et al., 1993;Saville and Houslay, 1994). Since this phosphorylation process occurs in intact

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Section: Discussionsupporting

confidence: 75%

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Benguría

Hernández‐Perera

Martínez‐Pastor

et al. 1994

European Journal of Biochemistry

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“…SDS gel electrophoresis in the presence of EGTA [22] (fig.2) caused a small but significant decrease in the mobility of the 22 kDa protein. The shift which occurs is less than that obtained with calmodulin which has 4 calcium-binding sites but is comparable to that of myosin light chain 2 which has only one.…”

Section: Resultsmentioning

confidence: 96%

Identification of a novel calcium‐binding protein (CP₂₂) in multidrug‐resistant murine and hamster cells

et al. 1986

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Analysis of cytoplasmic extracts of multidrug-resistant murine and hamster cells by SDS gel and 2D gel electrophoresis showed that they expressed an abundant 22 kDa protein which was absent from the drug sensitive parent lines. SDS gel electrophoresis in the presence of EGTA and direct binding tests with 45Ca2+ showed that the resistance-associated protein is a specific calcium-binding protein. Thus the development of multidrug resistance in both coic~cine-blats hamster cells and ad~amycin-sel~ted murine cells is associated with a major change in calcium metabolism. These observations provide the first molecular basis for the hypothesis that Ca2+ plays a central role in the development of the multidrug resistance phenomenon.

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“…The radioactive bands were detected with Kodak x-ray film. For the Ca 2ϩ -dependent electrophoretic mobility-shift assay, Ca 2ϩ -binding proteins were separated by SDS͞PAGE (12%) in the presence of 2 mM CaCl 2 or 2 mM EGTA (19).…”

Section: Methodsmentioning

confidence: 99%

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Zhao

Shi

Zhao

et al. 2005

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

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Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca 2؉ -binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. It has been demonstrated that [Ca 2ϩ ] i is tightly regulated in some bacteria (6), and there is evidence indicating that calcium is critical to some bacterial cellular processes such as sporulation of Bacillus (7), chemotaxis of Escherichia coli (8), and heterocyst differentiation of cyanobacteria (9). Heterocyst development may represent one of the earliest examples of pattern formation in evolution (10). Heterocysts are specialized cells for nitrogen fixation of some filamentous cyanobacteria (11)(12)(13)(14). When heterocystous cyanobacteria are subjected to nitrogen step-down, some vegetative cells differentiate and become heterocysts. Many genes are specifically involved in heterocyst differentiation. The genes that are required for initiation of cell differentiation include ntcA (15, 16) and hetR (17). HetR is a serine-type protease that plays an important role in heterocyst formation (18).Evidence obtained by manipulating extracellular calcium concentration and by using inhibitors of Ca 2ϩ -binding proteins suggested that Ca 2ϩ could be involved in heterocyst differentiation (9). Here we describe a Ca 2ϩ -binding protein, CcbP, from Anabaena sp. PCC 7120. Our study shows that free calcium accumulates in differentiating cells and mature heterocysts, correlated with a drop in the level of the Ca 2ϩ -binding protein.The free Ca 2ϩ concentration appears to be critical for the differentiation process. Materials and MethodsThe following protocols can be found in Supporting Text, which is published as supporting information on the PNAS web site: growth of the strains of Anabaena; isolation of the Ca 2ϩ -binding protein CcbP; isolation of the ccbP gene from total genomic DNA; expression of recombinant ccbP in Escherichia coli; construction of plasmids for inactivation of the ccbP gene, for copper-regulated expression of ccbP, for copper-regulated expression of rat calmodulin (CaM) gene, for ccbP promoter regulation of GFP expression, and for expression of obelin, a reporter for the level of free Ca 2ϩ .Assays for Ca 2؉ -Binding Proteins. 45 Ca 2ϩ overlay assay was performed as follows: Proteins were first separated by SDS͞PAGE and then transferred to a poly(vinylidene difluoride) membrane. It was then washed three times with buffer C (20 mM Tris⅐HCl, pH 7.2͞5 mM MgCl 2 ͞60 mM KCl͞10 mM imidazole). The memb...

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Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate

Cited by 261 publications

References 20 publications

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Identification of a novel calcium‐binding protein (CP₂₂) in multidrug‐resistant murine and hamster cells

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

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Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate

Cited by 261 publications

References 20 publications

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Identification of a novel calcium‐binding protein (CP22) in multidrug‐resistant murine and hamster cells

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

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Identification of a novel calcium‐binding protein (CP₂₂) in multidrug‐resistant murine and hamster cells