Calmodulin Concentration Research Articles

Calcium/calmodulin-dependent regulation of the NH 2-terminal F-actin binding domain of utrophin

The cytoskeletal proteins utrophin, dystrophin and α-actinin are predicted to form antiparallel dimers thus potentially bringing their NH 2-terminal F-actin binding domains in close proximity to their EF-hand containing COOH-terminal domains. This arrangement would allow for calcium-dependent regulation of F-actin binding. We tested this hypothesis by determining the effect of the ubiquitous calcium binding protein calmodulin on their F-actin binding capabilities. Binding of the NH 2-terminal F-actin binding domain of utrophin to F-actin was inhibited by increasing concentrations of calmodulin in a calcium-dependent manner. The homologous F-actin binding domains from dystrophin and α-actinin were not regulated by calmodulin in the presence or absence of calcium. These findings have implications for the structural organisation of utrophin dimers and also for the replacement of dystrophin by over-expression of utrophin in dystrophic muscle.

The C-terminal Half-molecular Domain of Calmodulin is Responsible for High-affinity Interaction with Target Enzymes.

Chimeric proteins from chicken and yeast calmodulin were prepared, and roles of three structural domains, N-domain (1-72), central helix (73-87) and C-domain (88-148), were evaluated. Mutants with the chicken-type C-domain activated the cyclic nucleotide phosphodiesterase with small values of Kact (the concentration of calmodulin giving a half maximal activation), a property of chicken calmodulin. On the other hand mutants with the yeast-type C-domain activated the phosphodiesterase with μM range of Kact, a property of yeast calmodulin. In activation of myosin light chain kinase, introduction of the yeast-type C-domain into chicken calmodulin increased the Kact value by more than 1000-fold with a dramatic decrease in the maximum activity (Vmax). On the other hand introduction of the chicken-type C-domain led to a profile with lower Kact and higher Vmax. Minor effects on Vmax and Kact were observed by substitution of the central helix. Although various small contributions of the N-domain were observed, a common role of the chicken-type C-domain was suggested to catch and maintain the high-affinity interaction with target enzymes.

Calmodulin regulates fast axonal transport of squid axoplasm organelles

The role of calmodulin (CaM) in organelle motility (fast axonal transport) in the axoplasm of the squid giant axon was evaluated directly using video-enhanced microscopy. Addition of 6 μM CaM to extruded squid axoplasm produced a 2.6-fold increase in the number of organelles moving per minute per unit area of axoplasm. When lower concentrations of CaM, including physiological concentration (2 μg/ml), were added to extruded axoplasm, the number of organelles moving was equally increased. CaM had no significant effect on the mean velocity of organelle translocations. The stimulatory effect of CaM was reduced significantly by the CaM inhibitors melittin (36 μM) and trifluoperazine (50 μM). Parvalbumin, a high-affinity calcium binding protein, did not stimulate motile activity. These results suggest that CaM is a positive regulator of fast axonal transport. At the molecular level, this regulation may involve microtubule-and/or actin-based motor proteins. Several possible molecular mechanisms are proposed.

Changes of calmodulin concentration and cyclic 3′, 5′-nucleotide phosphodiesterase activities in cardiac muscle of hyper- and hypothyroid rats

To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3',5'-nucleotide metabolism by measuring cyclic 3',5'-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats. Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats. It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was significantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca2+ metabolism.

Protein kinase C regulates calmodulin expression in NRK cells activated to proliferate from quiescence

We have investigated the levels of calmodulin protein and calmodulin mRNA species during prliferative activation of NRK cells. Cells activated to proliferate from quiescence started to replicate DNA at 15 h, reaching a maximum at 20 h after serum addition. The maximum of mitosis was observed at 24 h. Quiescent cells showed a calmodulin concentration of 1.5 ng/μg of protein. At 10 h after serum addition the amount of calmodulin started to increase, reaching values of 3.0 ng/μg of protein at 24 h. NRK cells expressed predominantly 3 species of calmodulin transcripts: the 1.7 kb from CaM I, the 1.4 kb from CaM II and the 2.3 kb from CaM III. The amount of all the 3 transcripts was low in quiescent cells and 10 h after activation the levels were already high, reaching a maximum around 20 h. At the latter time the amount of the 3 calmodulin mRNAs was 5–10-fold higher than in serum starved cells. Run-on experiments showed that at 20 h after activation the transcription rates of the 3 calmodulin genes were higher than in quiescent cells. The addition of protein kinase C inhibitors to the cultures blocked the increase of the calmodulin transcripts while inhibitors of protein kinase A did not have any effect. Moreover, the addition of submitogenic doses of phorbol 12-tetradecanoate induced the increase of all 3 calmodulin transcripts. These results indicate that protein kinase C regulates calmodulin expression when NRK cells are activated to proliferate.

Overexpression of the myristoylated alanine-rich C-kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists.

The acidic 80-kDa myristoylated alanine-rich C-kinase substrate protein (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80-kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1-80K, expressed 4.5 +/- 0.8-fold and 9.5 +/- 1.5-fold higher levels of 80-kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern-blot and Northern-blot analyses of Rat1-80K showed intact integration and correct transcription of the introduced 80-kDa MARCKS gene. The overexpressed 80-kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80-kDa MARCKS has been described as a calmodulin-binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and triflouperazine on the entry into the S-phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K cells was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that overexpression of the 80-kDa MARCKS protein reduces the free concentration of calmodulin in the cell.

Calmodulin Expression During Rat Liver Regeneration

We have investigated the messenger RNAs expressed from the three calmodulin genes during rat liver regeneration. The results revealed that all the calmodulin transcripts increased from 8 hr after a partial hepatectomy, although differences in the timing and the level of expression from the three genes were observed. Calmodulin I transcripts peaked at 16 hr, whereas calmodulin II and calmodulin III progressively increased from 8 to 24 hr. At 24 hr after surgery, calmodulin I, calmodulin II and the 2.3 kb calmodulin III transcripts reached values of a 6-fold increase, whereas the 0.8 kb product of calmodulin III increased 25-fold. At 30 hr the levels of all the calmodulin transcripts were similar to those observed at 24 hr. The transcription rates of the three calmodulin genes augmented after hepatectomy (calmodulin I and calmodulin II twofold and calmodulin III fourfold), indicating that the elevation of the calmodulin transcripts could be, at least partially, the result of this increase in the transcription rates. The total calmodulin concentration also increased twofold at 24 hr after hepatectomy. We also report that the administration of the beta-adrenergic blocker, D,L-propranolol inhibited the accumulation of calmodulin protein without significantly affecting the increase of the messenger RNAs. These results indicate that the expression of calmodulin observed during liver regeneration could be regulated by cyclic AMP at the translational or posttranslational level.

Calmodulin regulates nucleotide hydrolysis activity of tissue transglutaminase.

The interaction of calmodulin with purified guinea pig liver transglutaminase was studied. The nucleotide (ATP and GTP) hydrolysis activity of this tissue transglutaminase was transiently increased and then gradually decreased depending on calmodulin concentration. The peak activation was obtained in the presence of a stoichiometric amount of calmodulin. The effect of calmodulin on the classical transglutaminase activity was minimal. Fluorescence spectroscopy demonstrated that the enzyme produced a significant blue shift in the emission peak of dansylated calmodulin. Interestingly, Ca2+ was not required for the interaction between the two proteins. The results described here give an additional regulatory role to calmodulin.

Mutants of Smooth Muscle Myosin Light Chain Kinase at Tryptophan 800

The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.

Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion.

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca2+] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca2+, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca2+]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 microM and 90% at 7 microM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 microM trifluoperazine. Quantitative immunofluorescence using fluorescein-labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3-day bone-attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9 nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP-dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent Km for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated.

Inhibition of calmodulin-dependent myosin light-chain kinase by growth-hormone-releasing factor and vasoactive intestinal peptide.

In view of the ability of calmodulin to bind vasoactive intestinal peptide (VIP) and growth-hormone-releasing factor (GRF) with high affinity [Stallwood, Brugger, Baggenstoss, Stemmer, Shiraga, Landers and Paul (1992) J. Biol. Chem. 267, 19617-19621], the effects of these neuropeptides on a model calmodulin-dependent enzyme, myosin light-chain kinase (MLCK), were studied. Both peptides were potent inhibitors of MLCK activity. The inhibition of enzyme activity by VIP and GRF was progressively overcome with increasing calmodulin concentrations, with no inhibition observed at a saturating calmodulin concentration. Nanomolar concentrations of MLCK blocked the formation of calmodulin-[125I-Tyr10]VIP complexes. These data provide support for a functional role of VIP and GRF binding by calmodulin.

Expression of calmodulin and calmodulin binding proteins in lymphoblastoid cells

Calmodulin is encoded in vertebrates by three different genes: CALM1, CALM2, and CALM3. We have examined the mRNAs expressed from these three genes in eight lines of human lymphoblastoid cells (Namalwa, Raji, Ramos, JY, Molt-4, Jurkat, CEM, and HPB-ALL). We found that all these cell lines (except Ramos) overexpressed CALM3 transcripts, which led to an increase of total CaM protein with respect to quiescent normal T lymphocytes. The nuclear concentration of calmodulin was measured in two of these lymphoblastoid cell lines (JY and HPB-ALL) and compared to quiescent and phytohemagglutinin-activated T lymphocytes. Activated lymphocytes showed a 2-fold increase of nuclear calmodulin with respect to quiescent cells, whereas in the two lymphoblastoid cell lines, nuclear calmodulin remained similar to that of quiescent cells. The levels of a calmodulin-binding protein of 150 kDa in the homogenates of the eight lymphoblastoid lines was found to be higher than those of quiescent and activated lymphocytes. Likewise, the amount of three calmodulin-binding proteins of 240, 200, and 170 kDa was also increased in several of the cell lines, but not in all of them. The 170-kDa protein was only expressed by activated lymphocytes and lymphoblastoid cells, suggesting that it could be specific for proliferating cells. In the nuclei of activated lymphocytes and lymphoblastoid cells, a decrease of a calmodulin-binding protein of 110 kDa and increases of three other of 240, 180 and 170 kDa were also detected.

Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes

Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.

Regulation of the erythrocyte Ca2+-ATPase at high pH

The activation of the Ca(2+)-ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half-maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concentrations when the pH is increased. In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.

Raised urinary calmodulin levels in idiopathic myelofibrosis: possible implications for the aetiology of fibrosis

Platelet growth factors (e.g. PDGF and TGF-beta) are thought to be pathogenetically important in the stromal reaction characteristic of idiopathic myelofibrosis (IM). We have investigated a possible pathogenetic role for a further platelet mitogen, calmodulin. Platelets are rich in calmodulin, of which 30-40% is releasable with a time course that differs from alpha-granule proteins. In IM urinary calmodulin concentrations were 3-fold those of the normals controls. We suggest that an abnormal release of calmodulin may occur from platelets/megakaryocytes in patients with IM, and that calmodulin should be considered, along with other growth factors, in the pathogenesis of marrow fibrosis.

Most Egg Calmodulin Is a Follicle Cell Contribution to the Cytoplasm of the Blattella germanica Oocyte

A high concentration of calmodulin (CaM) appears in mid- to late vitellogenic cockroach follicles which composes 1.5% of total protein. CaM levels during oogenesis were estimated by densitometric analysis of immunoblots using anti- Blattella germanica egg CaM antibody as a probe. CaM accumulates in the follicle throughout the yolk deposition phase maintaining its highest level of accumulation during the later 6-fold increase in oocyte volume. Evidence suggests that this later accumulated CaM is synthesized by the follicle cells and deposited in the oocyte. In vitro experiments with [ 35S]-met showed that the highly abundant CaM accumulating in vitellogenic follicles may not all be synthesized by the oocyte. Isolated follicle cells incorporate 13-fold more [ 35S]-met into CaM than the oocytes themselves but do not accumulate the product. The follicle cells are capable of producing all the CaM observed in newly ovulated eggs. No CaM is detectable in transit in the hemolymph of the female. These facts argue that CaM produced by follicle cells is the most likely source of CaM in the vitellogenic oocyte. Indirect immunofluorescent staining with anti-egg CaM demonstrated that in early- and midvitellogenic follicles CaM is localized in the cytoplasm of follicle cells and the cytoplasmic compartment surrounding yolk granules of oocytes but is excluded from yolk granules. Immunofluorescence was most intense in the cortex of the oocyte and outside the membranes of yolk granules. Transport of CaM into the cytoplasmic compartment of the oocyte is possible without invoking traditional adsorptive endocytosis.

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth inNicotiana tabacumL.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

MS-282a and MS-282b, new inhibitors of calmodulin-activated myosin light chain kinase from Streptomyces tauricus ATCC 27470.

MS-282a and MS-282b were isolated from the culture broth of Streptomyces tauricus ATCC 27470 as inhibitors of smooth muscle myosin light chain kinase (MLCK). MS-282a and MS-282b inhibited the activity of chicken gizzard MLCK with IC50 values of 3.8 microM and 5.2 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited by 150 microM MS-282a at all. It is likely that MS-282a blocks MLCK activity by antagonizing calmodulin since 1) the compound inhibited calmodulin-dependent but not calmodulin-independent activity of MLCK; 2) the inhibition of MLCK was antagonized by increasing concentrations of calmodulin, and 3) the compound inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase.

The Ca2+ affinity of the plasma membrane Ca2+ pump is controlled by alternative splicing.

The plasma membrane Ca2+ pump is a calmodulin-regulated P-type ATPase that is an essential element in controlling intracellular Ca2+ concentration. Studies on the gene structure of this pump have revealed an alternate splice option that changes the structure of the calmodulin-binding domain. This change in the structure of the enzyme results in a reduced calmodulin affinity. Tests of the enzyme's activity in the presence of a high calmodulin concentration, approximating that found inside living cells, show that this reduced calmodulin affinity causes a reduced apparent affinity of the enzyme for Ca2+. This shift in the Ca2+ activation occurs in a Ca2+ concentration range crucial to cellular function and is probably the physiologically important consequence of the alternate splice.

Calmodulin and the regulation of smooth muscle contraction.

Calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein, mediates many of the regulatory effects of Ca2+, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca2+]i transient via the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca2+/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca2+. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca2+/calmodulin or indirectly by phosphorylation catalysed by Ca2+/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca2+ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca2+/calmodulin, e.g. the sarcolemmal Ca2+ pump and the ryanodine receptor/Ca2+ release channel, and other proteins which indirectly regulate [Ca2+]i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.