Intracellular Calmodulin Levels Research Articles

Calmodulin levels are dynamically regulated in living vascular smooth muscle cells.

The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescein isothiocyanate. Increases in NBD-GS17C fluorescence were detected by using confocal microscopy when chemically loaded cells were stimulated with solutions of elevated [K(+)] or the calcium ionophore 4-bromoA-23187 to elicit increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbol ester in the absence of changes in [Ca(2+)](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results suggest that the total unbound intracellular calmodulin levels may be sufficient to regulate the activity of caldesmon and, furthermore, that phosphorylation of protein kinase C substrates may increase the level of available calmodulin in living smooth muscle cells.

Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity.

Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes. Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels. However, little is known about the CaM-regulated enzymes involved. Protein phosphorylation has been shown to be important for cell-cycle regulation. Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle. As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe. The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2. Arrest was associated with a failure to activate the p34/cdc2 protein kinase. Expression of the mutant CaMKII in strains of S. pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.

Essential roles for calcium and calmodulin in G2/M progression in Aspergillus nidulans.

nimT encodes a protein in Aspergillus nidulans that is required for tyrosine dephosphorylation of p34cdc2 and has a strong homology to cdc25-type proteins. Conditional mutation of nimT (nimT23 mutation) arrests cells in G2 at the restrictive temperature. After release of the temperature-sensitive nimT23 block, p34cdc2 undergoes tyrosine dephosphorylation and we showed that as cells entered mitosis, a rapid increase in calmodulin was observed. The increase in calmodulin and progression into mitosis were prevented by reducing extracellular Ca2+ levels to 2 nM. The calmodulin gene of a nimT23-containing strain was replaced with a hybrid gene in which calmodulin transcription was regulated by the alcA promoter (AlcCaM/T23). This allowed experimental manipulation of the level of intracellular calmodulin by the carbon source in the medium. When either extracellular Ca2+ or intracellular calmodulin levels were reduced at the nimT23 G2 arrest point, p34cdc2 remained tyrosine phosphorylated but the mitotic NIMA kinase encoded by nimA was not activated. Release of the temperature sensitive nimT23 arrest when either extracellular Ca2+ or calmodulin concentrations were low blocked tyrosine dephosphorylation of p34cdc2, activation of NIMA and progression of cells into mitosis. However, reduced levels of either Ca2+ or calmodulin had no effect on the increase in histone H1 kinase activity associated with p13 beads or the degree of phosphorylation of the majority of MPM-2-reacting proteins following release of the nimT23 mutation. These results demonstrate that both Ca2+ and calmodulin are important for progression into mitosis from the nimT23 arrest point in a pathway involving activation of both NIMA and p34cdc2 protein kinases.

Calmodulin and Calmodulin-Binding Proteins during Cell Fusion in Dictyostelium Discoideum: Developmental Regulation by Calcium Ions

The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with Mrs of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, cross-linking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342, rabbit anti calmodulin antibody, FITC-labeled goat anti rabbit IgG and SR101 (sulfo rhodamine 101) simultaneously to stain individual normal and transformed cells, the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus, calmodulin and total protein respectively, could be recognized in individualj cells without interference. The phase of the cell cycle was determined by DNA content (Hoechst 33342). We found that in transformed cells (NIH3T3) tsRSV-LA90, cultured at 33°C and transformed C3H10T1/2 cells), the ratio of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV−LA90 cells, cultured at 39°C and C3H10T1/2 cells)in every cell cycle phase. This ratio increased obviously only from G1 to S phase in either normal or transformed cells. The results showed that calmodulin really increased during the transformation, and its increase was specific. In the meantime when cells proceeded from G1 to S, the intracellular calmodulin content also increased specifically.

Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication

In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin-binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin-binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin-binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin-binding protein, along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin-binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division.

The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12-15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12 h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.

48] Methods for analyzing bovine papilloma virus-based calmodulin expression vectors

Publisher Summary This chapter describes a method for the transfection and analysis of bovine papilloma virus (BPV) expression vector-containing cell lines. This method allows a rapid and simple way to identify cells that express calmodulin (CaM) gene. The approach used is generally applicable to the study of any BPV-based expression vector. Three different BPV-based CaM expression vectors were used to study the effect of changes in intracellular CaM levels on cellular function. The vectors BPV-CM and BPV-MCM II both contain a CaM minigene whose expression is regulated either by a CaM promoter or by the human metallothionein IIa promoter. Cells that retained unrearranged episomal plasmids are screened for expression of vector-derived mRNA by slot-blot and Northern blot analysis. Finally, cells are shown to be producing significantly higher levels of intact protein by techniques such as RIA or SDS-PAGE. The use of expression vectors has many applications in studying the biology of gene products in vivo. The synthesis of mutated proteins or the production of antisense RNA as a specific antagonist for a single gene product both hold promise in identifying physiologically relevant roles in systems where in vitro analysis has suggested an important function.

Binding of calmodulin to the neuronal cytoskeletal protein kinase type II cooperatively stimulates autophosphorylation.

The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.

Calmodulin levels in hypertensive rats

Intracellular calmodulin levels were measured by direct radioimmunoassay in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Decreased calmodulin levels were demonstrated in the brain, heart, aorta and kidney of spontaneously hypertensive rats compared with those in Wistar-Kyoto rats. Calmodulin levels in the brain were also decreased in deoxycorticosterone acetate (DOCA)-salt rats, but not changed significantly in the heart, aorta and kidney compared with those in Wistar-Kyoto rats. These findings suggest that intracellular calcium-dependent regulatory systems are genetically disrupted in spontaneously hypertensive rats, but this is probably not an important factor in the development of hypertension.

Calmodulin level and cAMP-dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogenesis

The changes in intracellular calmodulin levels and cAMP-dependent protein kinase activities have been studied in the testis of normally developing and hypophysectiomized rats. It appears that the onset of spermatogenesis which occurs on the first days of the postnatal development, is associated with a major (over fivefold) increase in the calmodulin level and enhancement of the cAMP-dependent protein kinase activity. On the contrary, hypophysectomy of adult animals is associated with a progressive decline in the calmodulin level and a rapid and regular decrease in the cAMP-dependent protein kinase activity. Moreover, measurements of the intracellular calmodulin level and cAMP-dependent kinase activity of isolated testicular germ cells or epididymal spermatozoa have shown that testosterone, administrated to hypophysectomized rats as subcutaneous implants, maintains the concentration of these regulatory proteins to normal values.

Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts.

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

Biosynthesis of Calmodulin in Normal and Virus-Transformed Chicken Embryo Fibroblasts

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

Regulation of intracellular levels of calmodulin and tubulin in normal and transformed cells.

Transformation of mammalian tissue culture cells by oncogenic viruses results in a 2-fold increase in the intracellular concentration of calmodulin quantitated by radioimmunoassay. The two pairs of companion cell lines used in this study were the Swiss mouse 3T3/simian virus 40-transformed 3T3 cells and the normal rat kidney (NRK)/Rous sarcoma virus-transformed NRK cells. The increased intracellular levels of calmodulin in the transformed cells are due to a greater increase in the rate of synthesis (3-fold) relative to the change in the rate of degradation (1.4-fold). On the other hand, no increases were observed in tubulin levels as quantitated by a colchicine-binding assay. The lack of change in tubulin concentration was accounted for by a 2-fold increase in the rate of degradation that is compensated by a similar increase in the rate of synthesis. The consequence of such changes in both transformed cell types is a 2-fold increase in the calmodulin-to-tubulin protein ratio relative to that in their nontransformed counterparts.