Calcitonin gene expression in the rat uterus during pregnancy.
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Animals↗
Biomedical subjects
I C Bagchi
Publications and source records attributed to I C Bagchi.
Ferritin heavy chain is a progesterone-inducible marker in the uterus during pregnancy.
The female sex steroid, progesterone, plays a central role in mammalian pregnancy by regulating crucial events in the uterus such as transformation of endometrium for implantation and maintenance of pregnancy. The hormone acts through its specific nuclear receptor and modulates the functions of target cells by controlling the synthesis of specific proteins. The identity of genes that are regulated by progesterone in the uterus during various phases of pregnancy, however, remains largely unknown. In this study, we employed a differential gene-screening method to identify the gene encoding ferritin heavy chain (FHC), a component of the multisubunit iron-binding protein ferritin, as being regulated by progesterone in the uterus. We observed that uterine expression of the FHC messenger RNAs (mRNAs) rose dramatically at the onset of pregnancy, coincident with the surge of progesterone. FHC expression continued at this elevated level throughout gestation when the progesterone concentration remained high. At term, FHC expression declined sharply as the progesterone concentration dropped. We localized FHC proteins exclusively in uterine stromal cells, a major site of action of progesterone during pregnancy. Administration of mifepristone, an antiprogestin, during the early stages of pregnancy abolished both FHC mRNA and protein expression, clearly suggesting a primary role of progesterone in the regulation of this gene. Consistent with this scenario, administration of progesterone to ovariectomized animals after a brief estrogen priming led to a marked (25-fold) induction of FHC mRNA in the uterus, whereas estrogen, dexamethasone, or dihydrotestosterone had no effect. Based on these results, we propose that FHC is a novel and useful marker to study progesterone-regulated events in the uterus during pregnancy.
Animals↗
Progesterone stimulates calcitonin gene expression in the uterus during implantation.
Implantation of the mammalian embryo into the wall of the uterus is regulated by a timely interplay of the ovarian hormones, estrogen and progesterone. These hormones orchestrate a set of modifications in the uterine endometrium that transforms it from a nonreceptive to a receptive phase allowing the implantation of the developing blastocyst. The molecular and cellular mechanisms underlying this complex process, however, remain largely unknown. To investigate the endocrine basis of uterine receptivity, we employed a gene expression screen technique to identify factors whose expressions are modulated in the rat uterus in response to estrogen and progesterone at the onset of implantation. Here we report that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is markedly enhanced in the uterus during pregnancy. By Northern blot analysis, we show that the synthesis of calcitonin messenger RNA is induced at the time of implantation. Immunocytochemistry with calcitonin antibody demonstrates further that the peptide is localized in the glandular epithelial cells of the uterus. The antiprogestin drug RU486, which is known to block implantation, abolishes calcitonin expression, suggesting a regulatory role for progesterone in this process. Consistent with this observation, progesterone significantly stimulates calcitonin messenger RNA and protein synthesis in the uteri of ovariectomized animals. Our study, therefore, identifies calcitonin as a stage- and cell-specific marker of progesterone action in the uterus during pregnancy. Estrogen exhibits no significant effect on calcitonin expression when administered alone to ovariectomized animals. However, a low dose of estrogen synergizes with progesterone, and a high dose antagonizes progesterone-mediated gene induction. Both estrogen and progesterone, therefore, modulate calcitonin gene expression in the uterus. The stage-specific regulation of calcitonin is apparently determined by the relative concentrations and the sequences of appearance of these two hormones and possibly other as yet unknown regulatory factors during pregnancy. We propose that calcitonin, a known regulator of calcium levels in the bone and kidney, may play an important regulatory role in the uterus of pregnant animals during the early events leading to implantation of the embryo.
Animals↗
Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.
Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.
Amino Acid Sequence↗
Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.
We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.
Amino Acid Sequence↗
Regulation of smooth muscle myosin light chain kinase by calmodulin.
The mutagenesis work described in this paper has been instrumental in furthering our understanding of how CaM binds to and activates MLCK. Figure 2 schematically represents this interaction. The inactive MLCK appears to have a catalytic domain that is repressed by a substrate inhibitory domain that overlaps with the CaM binding domain, a basic amphipathic helix. In the presence of Ca2+, CaM undergoes a conformational change that exposes two hydrophobic pockets, one in each globular lobe, that are important for binding to MLCK. Upon binding CaM, MLCK undergoes a conformational change that derepresses the catalytic site, allows substrate access and light chain phosphorylation. Calmodulin antagonist drugs intercalate within these hydrophobic pockets to interfere with target enzyme binding. The total loss of activity if W800 is altered to A illustrates the importance of these hydrophobic interactions within the enzyme. The basic residues are also important; most of the basic residues in the binding domain of MLCK appear to aid in CaM binding but are not in themselves crucial, this includes the RRK triad. However, a specific electrostatic interaction between R812 of MLCK and CaM is suggested by the complete failure in MLCK activation if this residue is changed to an A. Electrostatic interactions between MLCK and CaM are also indicated by the TaM-BM1 mutant. This mutant can bind to but not activate MLCK. It is hypothesized that TaM-BM1 will bind to the basic amphipathic helix of MLCK but that the alterations in the surface charges (especially E14 and T34) and/or hydrophobicity (S38) prevent the proper conformational change in MLCK necessary for light chain phosphorylation. The resulting MLCK-CaM complex is therefore, inactive but can bind TaM-BM1. The exact interaction of these amino acids in CaM with MLCK will have to await the elucidation of a CaM-MLCK co-crystal.
Amino Acid Sequence↗
Myosin light chain kinase structure function analysis using bacterial expression.
A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.
Animals↗
Molecular analysis of calmodulin and smooth muscle myosin light chain kinase.
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3',5'-Cyclic-GMP Phosphodiesterases↗
The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.
Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.
Amino Acid Sequence↗