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PubMed1989

Phenothiazine-binding and attachment sites of CAPP1-calmodulin.

In the presence of Ca2+ norchlorpromazine isothiocyanate forms a monocovalent complex with calmodulin: CAPP1-calmodulin (Newton et al, 1983). Trypsin digestion of [3H]CAPP1-calmodulin yields as the major radioactive peptide N epsilon-CAPP-Lys-Met-Lys, corresponding to residues 75-77 of calmodulin. Stoichiometric amounts of all other expected tryptic peptides are also found, indicating that norchlorpromazine isothiocyanate selectively acylates Lys 75. A second molecule of CAPP-NCS can react, albeit slowly, with calmodulin to form CAPP2-calmodulin. Fragments 38-74 and 127-148 are completely missing from the trypsin digests of CAPP2-calmodulin without deliberate exposure to UV irradiation. Possibly the lengthy preparation of CAPP2-calmodulin favors photolysis, caused by room lights, of the putative CAPP-binding domains located in these two peptides. Lys 148, the sole lysyl residue in fragment 127-148, is a probable site of attachment of the second molecule of CAPP. UV irradiation of CAPP1-calmodulin, followed by digestion with trypsin, results in the selective loss of 50% each of peptides containing residues 38-74 and 127-148, suggesting that these peptides contain the hydrophobic amino acids that form the phenothiazine-binding sites. The loss of peptides encompassing residues 38-74 and 127-148, located in the amino and carboxyl halves of calmodulin, respectively, suggests that the hydrophobic rings of CAPP can bind at either one of the two phenothiazine sites. Computer modeling of CAPP1-calmodulin with the X-ray coordinates of calmodulin (Babu et al., 1986) indicates that CAPP attached to Lys 75 cannot interact with the carboxyl-terminal phenothiazine-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

The calmodulin content of the epidermis in psoriasis.

The calmodulin content of epidermis was determined by assay of biologically active and radioimmunoassayable calmodulin in epidermal biopsy samples of 16 normal control subjects and 36 patients with psoriasis. Calmodulin levels in the involved epidermis of patients with psoriasis were significantly greater than in epidermis of control subjects, with both methods of calmodulin measurement. Levels of calmodulin in the uninvolved epidermis were also elevated but to a lesser degree, achieving statistical significance only when measured by radioimmunoassay. However, the degree of correlation between the two measurements of calmodulin was poor for the patient samples, suggesting that each may measure a different form of calmodulin. The specificity of the elevated calmodulin in psoriatic epidermis was investigated by measuring calmodulin in another unrelated tissue. Calmodulin activity in circulating peripheral blood lymphocytes of seven patients with psoriasis was similar to that found in the lymphocytes of ten normal volunteers. The relationship between calmodulin and the hyperproliferative state of the psoriatic epidermis was investigated. No significant increase in calmodulin activity was found after mitogen stimulation of lymphocyte proliferation or after Sellotape-stripping of the epidermis by a protocol which has been shown to cause hyperproliferation of the epidermis.

Adult↗
PubMed1985

Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.

Calmodulin stimulated the rate of Mg2+-induced polymerization of human platelet actin. The stimulatory effect was due to an increase in the nucleation phase of the reaction; there was no effect on the steady-state viscosity. The calmodulin antagonist trifluoperazine blocked the stimulatory effect of calmodulin. Addition of EGTA to the reaction mixture also stimulated the rate of actin polymerization; however, the effect of calmodulin on actin polymerization is not due to Ca2+ chelation, as is presumed to be the case for EGTA. Electron microscopy revealed structural differences in the filaments prepared in the presence of calmodulin as compared to those prepared with trifluoperazine. In the presence of calmodulin, the filaments were thicker, suggesting that they consisted of multiple actin polymers. In addition, numerous projections were present perpendicular to the filaments, as well as localized areas of filament bundling. It was not possible to demonstrate a direct interaction between calmodulin and actin, which raises the possibility that the calmodulin effect may be indirect through a calmodulin-binding protein or calmodulin-dependent enzyme. Regardless of whether calmodulin is acting directly or indirectly, these results provide evidence that calmodulin may play a regulatory role in either the polymerization of actin or in determining the structural characteristics of actin filaments.

Actins↗
PubMed1986

Peptide antisera as sequence-specific probes of protein conformational transitions: calmodulin exhibits calcium-dependent changes in antigenicity.

Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by approximately equal to 40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and the antigenic reactivity of calmodulin for the peptide antisera in the absence of calcium, the regions of the protein monitored by these antisera are exposed to the surface in both conformational states and probably accessible to specific antibodies. The apparent preference of peptide antibodies for one conformation of the molecule suggests that changes in the conformation of calmodulin occur in cognate sequences that are transformed by calcium from antigenic, flexible structures to less antigenic, relatively helical structures. Peptide antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes providing the cognate sequence is sterically accessible to antibody in both states but antigenic in only one.

Amino Acid Sequence↗
PubMed1987

Human salivary gustin is a potent activator of calmodulin-dependent brain phosphodiesterase.

Human salivary gustin stimulated activity of brain calmodulin-dependent cyclic nucleotide phosphodiesterase (cAMP PDEase; 3',5'-cyclic-nucleotide phosphodiesterase, EC 3.1.4.17) in a dose-dependent manner in the absence of calmodulin. At physiological levels found in human saliva, gustin activated cAMP PDEase 5- to 6-fold. Activation of PDEase occurred with as little as 500 ng of gustin. Comparative sensitivity of activation of PDEase by gustin was intermediate between calmodulin and lysophosphatidylcholine with maximal activation and half-maximal activation (indicated in parentheses) at 3 X 10(-8) M (4.3 X 10(-9) M), 3.4 X 10(-6) M (3.4 X 10(-7) M), and 2.5 X 10(-3) M (4.0 X 10(-5) M) for calmodulin, gustin, and lysophosphatidylcholine, respectively. No other major salivary protein activated PDEase. Anticalmodulin antibody completely inhibited calmodulin-activated cAMP PDEase activity, but the antibody had no effect on gustin-activated cAMP PDEase activity. A sensitive calmodulin RIA indicated that no calmodulin was detected in any gustin preparation that activated cAMP PDEase. Both gustin and calmodulin rendered cAMP PDEase thermally labile to a similar extent and increased Vmax without affecting the apparent Km for the substrate cAMP. Activation by gustin and calmodulin was unaffected by lubrol-PX, trypsin inhibitor, pepstatin A, or leupeptin. In the presence of 1 mM EGTA, gustin activated cAMP PDE 5- to 6-fold, but the activating ability was completely lost after gustin was heated at 100 degrees C for 5 min. In contrast, calmodulin lost all activating ability in the presence of 1 mM EGTA, whereas heating calmodulin at 100 degrees C for 5 min did not affect its activation of cAMP PDEase. Lysophosphatidylcholine-activation of cAMP PDEase, like gustin activation, was unaffected by EGTA, but lysophosphatidylcholine-activation of cAMP PDEase, like calmodulin activation, was unaffected by heating at 100 degrees C for 5 min.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Ca(2+)-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells.

A key assumption of most models for calmodulin regulation of smooth and non-muscle contractility is that calmodulin is freely diffusible at resting intracellular concentrations of free Ca2+. However, fluorescence recovery after photobleaching (FRAP) measurements of three different fluorescent analogs of calmodulin in cultured bovine tracheal smooth muscle cells suggest that free calmodulin may be limiting in unstimulated cells. Thirty-seven % of microinjected calmodulin is immobile by FRAP and the fastest recovering component has an effective diffusion coefficient 7-fold slower than a dextran of equivalent size. Combining the FRAP data with extraction data reported in a previous paper (Tansey, M., Luby-Phelps, K., Kamm, K.E., and Stull, J.T. (1994) J. Biol. Chem. 269, 9912-9920), we estimate that at most 5% of total endogenous calmodulin in resting smooth muscle cells is unbound (freely diffusible). Examination of the Ca2+ dependence of calmodulin mobility in permeabilized cells reveals that binding persists even at intracellular Ca2+ concentrations as low as 17 nM. When Ca2+ is elevated to between 450 nM and 3 microM, some of the bound calmodulin is released, as indicated by an increase in the effective diffusion coefficient and the percent mobile fraction. At higher Ca2+, calmodulin becomes increasingly immobilized. In about 50% of the cell population, clamping Ca2+ at micromolar levels results in translocation of cytoplasmic calmodulin to the nucleus. The compartmentalization and complex dynamics of calmodulin in living smooth muscle cells have profound implications for understanding how calmodulin regulates contractility in response to extracellular signals.

Animals↗
PubMed2001

Calmodulin enhances the stability of the estrogen receptor.

The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to calmodulin-Sepharose in vitro. However, the association of endogenous calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5-10-fold the association of calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5-2-fold. In contrast, the structurally distinct calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type calmodulin, but not a mutant calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.

Benzimidazoles↗
PubMed2002

Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts.

We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345-7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts. In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13.

3T3 Cells↗
PubMed2005

Fas binding to calmodulin regulates apoptosis in osteoclasts.

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.

Animals↗
PubMed1986

Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation.

Flagellates of Naegleria gruberi contain two calmodulins that differ in apparent molecular weight and intracellular location. Calmodulin-1, localized in flagella, has an apparent molecular weight of approximately 16,000, approximately the size of other protozoan calmodulins, whereas calmodulin-2, localized in cell bodies, is 15,300. Both proteins, purified, are calmodulins by several criteria, including Ca2+-dependent stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase and affinity for antibodies to vertebrate calmodulin. The finding of two calmodulins is unusual. Since the only known difference is apparent molecular weight, one calmodulin could be derived from the other, except that both calmodulins are synthesized in a wheat germ, cell-free system directed by RNA from differentiating Naegleria. Translatable mRNAs encoding calmodulins 1 and 2, not detected in amebas, appear and subsequently disappear concurrently during the 100-min differentiation of Naegleria from amebas to flagellates. Furthermore, these mRNAs increase and then decrease in abundance concurrently with those for flagellar tubulins, which suggests the possibility that the expression of the unrelated genes for calmodulin and tubulin may be under coordinate control during differentiation.

Animals↗
PubMed1993

A calmodulin-like protein as an extracellular mitogen for the keratinocyte.

This study investigated the importance of extracellular calmodulin to the proliferation of the keratinocyte. Normal keratinocytes in culture produced a calmodulin-like protein in their culture media, the level of which increased abruptly and transiently during their growth. This protein was calmodulin-like, in that it specifically bound to a calmodulin affinity column, exhibited calmodulin-like immunoreactivity in both an ELISA and on immunoblots when immunostained with a monoclonal antibody against calmodulin, had an apparent M(r) between 18,000 and 20,000, and stimulated activity in a calmodulin-dependent phosphodiesterase enzyme assay. Addition of exogenous pure calmodulin was of no further mitogenic benefit to the keratinocytes, and slightly reduced proliferation under the culture conditions used. However, addition of either a neutralizing antibody to calmodulin, or W7-agarose, to the culture media of proliferating cells markedly inhibited their proliferation. Accordingly, a calmodulin-like protein was found to satisfy all but one of the criteria for its action as an autocrine growth factor for the keratinocyte. We propose that the lack of mitogenic response to calmodulin in vitro is due to the cell meeting its own requirement for extracellular calmodulin.

Biological Assay↗
PubMed1986

Posttranslational modification of calmodulin in rat brain and pituitary.

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.

Amino Acids↗
PubMed1988

Differential regulation by calmodulin of basal, GTP-, and dopamine-stimulated adenylate cyclase activities in bovine striatum.

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.

Adenylyl Cyclases↗
PubMed1994

Purification and characterization of Bordetella calmodulin-like protein.

Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca(2+)-dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca2+ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis.

Adenylyl Cyclases↗
PubMed1992

Calmodulin content and activity in normal and coeliac duodenum.

Calmodulin is an important modulator of intracellular calcium processes and may be implicated in the calcium malabsorption of coeliac disease. The calmodulin content in extracts of duodenal biopsy specimens from 48 normal control subjects and 28 patients with coeliac disease was determined. Radioimmunoassay was used to measure immunoreactive calmodulin while a cyclic adenosine 3',5'-monophosphate phosphodiesterase activity assay was used to measure biologically active calmodulin. Calmodulin values measured by both assays were similar for control and disease groups. Mean (SEM) immunoreactive calmodulin values were 1.68 (0.09) micrograms/mg protein for controls and 1.67 (0.15) and 1.45 (0.15) micrograms/mg protein for partial and total villous atrophy respectively. These values were not significantly different. Biologically active calmodulin values were 2.77 (0.21), 1.82 (0.34), and 3.24 (0.33) micrograms/mg protein for control, partial, and total villous atrophy subjects respectively. The biologically active calmodulin values in the partial villous atrophy group were significantly lower than in controls and total villous atrophy subjects. In the phosphodiesterase assay, the calmodulin antagonist trifluoperazine inhibited the activity stimulated by purified calmodulin and by the extracts to the same extent. These results show that calmodulin values are normal in coeliac disease and provide no evidence that changes in calmodulin account for the abnormal calcium absorption in these patients.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

Actins↗
PubMed1989

Calmodulin as substrate for insulin-receptor kinase. Phosphorylation by receptors from rat skeletal muscle.

Calmodulin is a substrate for insulin-receptor kinase obtained from rat adipocytes and hepatocytes and human placenta. In this study, we demonstrate that insulin stimulates the phosphorylation of calmodulin via insulin receptors partially purified from rat skeletal muscle. Phosphorylation of calmodulin was maximal in the presence of Mg2+ and insulin and the absence of Ca2+. Free-Ca2+ concentrations greater than 0.1 microM progressively inhibited phosphorylation with almost total inhibition at 200 microM Ca2+. Insulin-stimulated phosphorylation of calmodulin was dose dependent and saturable with half-maximal effect obtained at approximately 5 x 10(-10) M insulin. There was an absolute requirement for certain basic proteins, e.g., polylysine or protamine sulfate, to obtain phosphate incorporation into calmodulin. Polylysine stimulated the phosphorylation of calmodulin independently of insulin, but this was increased up to sixfold by the addition of insulin. Phosphate incorporation into calmodulin increased with increasing concentration of the substrate up to a saturating concentration of 2.4 microM. The Km for calmodulin was approximately 0.2 microM. Up to 0.15 mol of phosphate was incorporated per mole of calmodulin with tyrosine the predominant amino acid phosphorylated. The observations that calmodulin is phosphorylated by insulin-receptor kinase from all three classic target organs for insulin confirm that calmodulin is a general substrate for this kinase and suggest that Ca2+ and calmodulin may be components of the insulin-signaling mechanism.

Amino Acids↗
PubMed2002

Localization and regulation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase by cyclic AMP-dependent protein kinase.

Calmodulin-dependent cyclic nucleotide phosphodiesterase is one of the key enzymes involved in the complex interactions, which occur between the cyclic nucleotide and Ca2+ second-messenger systems. In eye, cAMP regulation is important in a variety of physiological processes such as aqueous humor regulation, photoreceptor signal transduction and retinal blood flow. Bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was purified to apparent homogeneity and the isolated enzyme had a significantly higher affinity for calmodulin and Ca2+. Immunohistology revealed calmodulin-dependent cyclic nucleotide phospho-diesterase expression in corneal epithelium, retina and optic nerve of the eye. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase and the following observations were made. Firstly, the phosphorylation resulted in the incorporation of 1 mol of phosphate per mol of subunit, resulting in higher calmodulin and Ca2+ concentration requirement for calmodulin-dependent cyclic nucleotide phosphodiesterase activation. Secondly, Ca2+ and calmodulin prevented the phosphorylation. Thirdly, the phosphorylation of calmodulin-dependent cyclic nucleotide phosphodiesterase could be reversed by the calmodulin-dependent phosphatase, calcineurin. Analysis of the complex regulatory properties of the calmodulin-dependent cyclic nucleotide phosphodiesterase in the eye has led to the suggestion that fluxes of cAMP and Ca2+ during cell activation are closely coupled and that calmodulin-dependent cyclic nucleotide phosphodiesterase plays a key role in this signal coupling phenomenon.

3',5'-Cyclic-AMP Phosphodiesterases↗