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Results for “regulatory module”

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PubMed1995

Hepatitis B virus transactivator protein X interacts with the TATA-binding protein.

Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX. The minimal region in pX sufficient for its interaction with TBP includes aa 110-143. Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX. The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP. These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters. This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.

Amino Acid Sequence↗

PubMed1997

Signaling in plants.

Higher plants are sessile organisms that perceive environmental cues such as light and chemical signals and respond by changing their morphologies. Signaling pathways utilize a complex network of interactions to orchestrate biochemical and physiological responses such as flowering, fruit ripening, germination, photosynthetic regulation, and shoot or root development. In this session, the mechanisms of signaling systems that trigger plant responses to light and to the gaseous hormone, ethylene, were discussed. These signals are first sensed by a receptor and transmitted to the nucleus by a complex network. A signal may be transmitted to the nucleus by any of several systems including GTP binding proteins (G proteins), which change activity upon GTP binding; protein kinase cascades, which sequentially phosphorylate and activate a series of proteins; and membrane ion channels, which change ionic characteristics of the cells. The signal is manifested in the nucleus as a change in the activity of DNA-binding proteins, which are transcription factors that specifically interact and modulate the regulatory regions of genes. Thus, detection of an environmental signal is transmitted through a transduction pathway, and changes in transcription factor activity may coordinate changes in the expression of a portfolio of genes to direct new developmental programs.

Arabidopsis↗

PubMed1998

T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70.

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.

Amino Acid Sequence↗

PubMed2000

Annexin 24 from Capsicum annuum. X-ray structure and biochemical characterization.

This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wild-type protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2. 8 A resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms.

Adenosine Triphosphatases↗

PubMed2000

SPECs, small binding proteins for Cdc42.

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.

3T3 Cells↗

PubMed2002

Cyclin D1 Is a Ligand-independent Co-repressor for Thyroid Hormone Receptors.

Thyroid hormone receptors (TRs) are critical regulators of growth, differentiation, and homeostasis. TRs function by regulating the expression of thyroid hormone (T3) target genes in both ligand-dependent and -independent pathways. Distinct classes of co-regulatory proteins modulate these two pathways. We show here a novel role of cyclin D1 as a T3-independent co-repressor for TRs. Cyclin D1 interacted with TR in vitro and in cells in a ligand-independent manner. Cyclin D1 acted to repress both the silencing activity of the unliganded TR and the transcriptional activity of the liganded TR. The repression was not due to the inhibition of the binding of TR to the thyroid hormone response element but by serving as a ligand-independent bridging factor to selectively recruit HDAC3 to form ternary complexes. The repression was augmented by increasing expression of HDAC3 but not by HDAC1 and was alleviated by trichostatin A. Thus, cyclin D1 is a novel ligand-independent co-repressor that opens a new paradigm to understand the molecular basis of the silencing action of TR.

Animals↗

PubMed2003

Magnesium ion-dependent activation of the RecA protein involves the C terminus.

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

Adenosine Triphosphate↗

PubMed2003

Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors.

Activation of dopamine D1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D1 receptors are co-localized with N-methyl-d-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D1 receptor complex was translocated to the plasma membrane. These data suggest that D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D1 receptor desensitization and cellular trafficking.

Animals↗

PubMed2003

p21Cip1 gene expression is modulated by Egr1: a novel regulatory mechanism involved in the resveratrol antiproliferative effect.

Epidemiological observations indicate that resveratrol, a natural antioxidant stilbene, exerts cardioprotective and chemopreventive effects. Moreover, the molecule induces in vitro cell growth inhibition and differentiation. Using human erythroleukemic K562 cells as model system, we demonstrated that resveratrol induces a remarkable gamma-globin synthesis, the erythroid differentiation being linked to impairment of cell proliferation, increased p21Cip1 expression and inhibition of cdk2 activity. The up-regulation of p21Cip1 transcription is prevented by cycloheximide, indicating the requirement of intermediate protein(s), which, in turn, regulate gene expression. The quantitative analysis of some transcription factors involved in the erythroid lineage, namely GATA-1, GATA-2, and Egr1, indicated that resveratrol selectively up-regulates Egr1 by an Erk1/2-dependent mechanism. The presence of an Egr1 consensus sequence in the p21Cip1 promoter suggested the hypothesis that this transcription factor directly regulates the expression of the cdk inhibitor. Transfection studies with deleted gene promoter constructs, as well as EMSA, pull-down, and chromatin immunoprecipitation experiments substantiated this view, demonstrating that Egr1 binds in vitro and in vivo to the identified consensus sequence of the p21Cip1 promoter. Moreover, an Egr1 phosphorothioate antisense hinders p21Cip1 accumulation and the antiproliferative effects of resveratrol. In conclusion, this is the first demonstration that Egr1 controls p21Cip1 expression by directly interacting with a specific sequence on its gene promoter. The identified regulatory mechanism also contributes to the clarification of the complex chemopreventive and antiproliferative properties of resveratrol.

Cell Differentiation↗

PubMed2004

Aryl hydrocarbon nuclear translocator (ARNT) promotes oxygen-independent stabilization of hypoxia-inducible factor-1alpha by modulating an Hsp90-dependent regulatory pathway.

Hypoxia-inducible factor-1 (HIF-1) is a potent cellular survival factor contributing to tumorigenesis in a broad range of cancers. The functional transcription factor exists as a heterodimeric complex consisting of HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Association of HIF-1 with ARNT is required for its activity; however, no other role has been ascribed to this interaction. We demonstrated previously that pharmacologic inhibition of Hsp90 by geldanamycin (GA) impairs HIF transcription and promotes VHL (Von Hippel-Lindau)-independent degradation of the protein, thus implicating Hsp90 as an essential interacting partner for HIF. In this study, we further explore the physiological role for Hsp90 in HIF function. We establish that the PAS (Per-ARNT-Sim) domain of HIF is required both to promote association with Hsp90 and confer sensitivity to GA. Coincidentally, this domain also associates with ARNT. Overexpression of ARNT in a VHL-deficient background resulted in substantially increased HIF-1 protein concomitant with increased protein stability. Conversely, down-regulation of endogenous ARNT protein by RNA interference decreased the steady-state HIF protein. ARNT-mediated stabilization of HIF is specific for the Hsp90-dependent pathway, as ARNT was unable to protect HIF from VHL-mediated degradation. We establish that the ability of ARNT to up-regulate HIF and diminish HIF sensitivity to GA is due to its ability to compete for the Hsp90 binding site on HIF. These data elucidate novel functions for ARNT and Hsp90 in regulating HIF function and further illustrate that cofactor association may significantly impact upon the sensitivity of Hsp90 clients to chaperone inhibitors.

Animals↗

PubMed2005

Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism.

Protein kinase A (PKA) has long been recognized as playing a major role in many regulatory processes in cells through its activation by the ubiquitous second messenger cAMP. We show here a novel mode of activation of PKA type II that is independent of cAMP and is, instead, dependent on sphingosine. PKA type II is specifically activated by sphingosine and its analog, dimethylsphingosine, but not by sphingosine-1-phosphate or other lipids. Like cAMP, sphingosine activates PKA holoenzyme but not the catalytic subunit alone, suggesting that the activation is mediated by the regulatory subunits. However, sphingosine-activated PKA, but not cAMP-activated PKA, is inhibited by phosphatidylserine, suggesting a distinct mechanism of activation. Furthermore, unlike cAMP, sphingosine does not induce the dissociation of PKA holoenzyme into catalytic and regulatory subunits. Modulation of sphingosine levels in vivo results in alteration in basal membrane-associated PKA activity consistent with a direct effect of membrane sphingosine on PKA type II. Importantly, sphingosine-dependent but not cAMP-dependent activation of PKA specifically phosphorylates Ser58 of the multifunctional adapter protein 14-3-3zeta, promoting the conversion of dimeric 14-3-3 to a monomeric state, thus potentially modulating several biological functions. These results define a new mode of PKA activation that is sphingosine-dependent and mechanistically different from the classical cAMP-dependent activation of PKA. Furthermore, they suggest that stimuli that induce sphingosine accumulation and modulate phospholipid content at the cell membrane have the potential to activate PKA, thereby inducing the phosphorylation of distinct substrates and biological activities.

14-3-3 Proteins↗

PubMed2001

Pro-inflammatory cytokines and adipose tissue.

Cytokines appear to be major regulators of adipose tissue metabolism. Therapeutic modulation of cytokine systems offers the possibility of major changes in adipose tissue behaviour. Cytokines within adipose tissue originate from adipocyte, preadipocyte and other cell types. mRNA expression studies show that adipocytes can synthesise both tumour necrosis factor alpha (TNF-alpha) and several interleukins (IL), notably IL-1beta and IL-6. Other adipocyte products with 'immunological' actions include complement system products and macrophage colony-stimulating factor. Cytokine secretion within adipocytes appears similar to that of other cells. There is general agreement that circulating TNF-alpha and IL-6 concentrations are mildly elevated in obesity. Most studies suggest increased TNF-alpha mRNA expression or secretion in vitro in adipose tissue from obese subjects. The factors regulating cytokine release within adipose tissue appear to include usual 'inflammatory' stimuli such as lipopolysaccaride, but also the size of the fat cells per se and catecholamines. There is conflicting data about whether insulin and cortisol regulate TNF-alpha. The effects of cytokines within adipose tissue include some actions that might be characterised as metabolic. TNF-alpha and IL-6 inhibit lipoprotein lipase, and TNF-alpha additionally stimulates hormone-sensitive lipase and induces uncoupling protein expression. TNF-alpha also down regulates insulin-stimulated glucose uptake via effects on glucose transporter 4, insulin receptor autophosphorylation and insulin receptor substrate-1. All these effects will tend to reduce lipid accumulation within adipose tissue. Other effects appear more 'trophic', and include the induction of apoptosis, regulation of cell size and induction of de-differentiation (the latter involving reduced peroxisome proliferator-activated receptor gamma). Cytokines are important stimulators and repressors of other cytokines. In addition, cytokines appear to modulate other regulatory systems. Examples of the latter include effects on leptin secretion (probably stimulation followed by inhibition) and reduction of beta3-adrenoceptor expression. There seems to be no clear agreement as to which cytokines derived from adipose tissue act as remote regulators, i.e. hormones. Leptin, which is structurally a cytokine, is also a hormone. IL-6 appears to be released systemically by adipose tissue, but TNF-alpha is probably not. Both leptin and IL-6 appear to act on the hypothalamus, IL-6 acts on the liver, while leptin may have actions on the pancreas. The importance of the immune system in whole-body energy balance provides a rationale for the links between cytokines and adipose tissue. It seems clear that TNF-alpha is a powerful autocrine and paracrine regulator of adipose tissue. Other cytokines, notably leptin, and possibly IL-6, have lesser actions on adipose tissue. These cytokines act as hormones, reporting the state of adipose tissue stores throughout the body.

Adipocytes↗

PubMed1999

Shc and FAK differentially regulate cell motility and directionality modulated by PTEN.

Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. We previously described inhibition of cell migration by PTEN and restoration of motility by focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130(Cas)). We now report a novel pathway regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulated integrin- mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibited cell migration; Akt appeared uninvolved. PTEN directly dephosphorylated Shc. The migration induced by FAK or p130(Cas) was directionally persistent and involved extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induced a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration.

Actins↗

PubMed1998

The thyrotropin-releasing hormone-degrading ectoenzyme: the third element of the thyrotropin-releasing hormone-signaling system.

The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits an extraordinary high degree of substrate specificity and other unusual characteristics. There is no other ectopeptidase known capable of degrading this tripeptideamide, and vice versa, TRH is the only known substrate of this unique enzyme. Thus, studies on this enzyme may reveal new aspects on the function of the TRH signaling system. After succeeding in purifying this enzyme to homogeneity and cloning the cDNA encoding rat TRH-DE, molecular tools became available to study the expression of this enzyme by Northern blot analysis and in situ hybridization histochemistry. The stringent and tissue-specific regulation of the adenohypophyseal TRH-DE by estradiol and thyroid hormones strongly suggests that this enzyme may act as a regulatory element modulating pituitary hormone secretion. In brain, the expression of TRH-DE is not influenced by peripheral hormones but the distinct distribution pattern, and the high activities support the concept that in this tissue TRH-DE may act as a terminator of TRH signals.

Aminopeptidases↗

PubMed1997

Dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver.

Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that coordinate cellular iron homeostasis in mammals. We investigated the effect of dietary iron intake on rat liver IRP activity in relation to the abundance of two targets of IRP action, ferritin and mitochondrial aconitase (m-aconitase). Rats were fed diets containing 2, 11, 20, 37 (control), 72 or 107 mg iron/kg diet for 3 wk. RNA binding activity of IRP1 and IRP2 was enhanced one- to twofold in rats fed 11 or 2 mg iron/kg diet compared with control rats. IRP RNA binding activity was inversely correlated to blood hemoglobin levels (r = -0.787; P < 0.0001). Compared with control rats, liver ferritin levels were depressed in rats fed 20 mg iron/kg diet and were undetectable in rats ingesting diets with 11 or 2 mg iron/kg diet. Ferritin concentrations were biphasically related to IRP RNA binding activity with the regulation of IRP occurring before the onset of ferritin accumulation. Iron deficiency caused up to a 50% decline in m-aconitase abundance. IRP RNA binding activity and m-aconitase abundance were inversely correlated (r = -0.751; P < 0.0001). Our results indicate that (1) liver IRP activity is responsive to a range of dietary iron levels, (2) there appears to be a differential effect of IRPs on ferritin and m-aconitase abundance, and (3) activation of IRPs may contribute to the alterations in energy metabolism in iron deficiency through an impairment of m-aconitase synthesis.

Aconitate Hydratase↗

PubMed1986

Nucleotide sequence of the Saccharomyces cerevisiae MET25 gene.

To elucidate further the molecular basis of the specific regulatory mechanism modulating the expression of the genes implicated in methionine metabolism, we have cloned and characterized two genes, MET3 and MET25, and shown that the regulation of their expression is transcriptional. The sequence of the cloned yeast MET25 gene which encodes the O-acetyl homoserine - O-acetyl serine (OAH-OAS) sulfhydrylase is reported here along with its 5' and 3' flanking regions. The amino acid composition predicted from the DNA sequence is in good agreement with that determined by hydrolysis of the purified enzyme. In the 5' flanking region the signal for general amino acid control was not found, corroborating our previous finding that the synthesis of OAH-OAS sulfhydrylase is not submitted to general control. The transcription start points have been determined. The 5' and 3' flanking regions of the MET25 gene suggest initiation and termination signals similar to those associated with other yeast genes.

Amino Acid Sequence↗

PubMed1998

Function of the C-terminal transactivation domain of human heat shock factor 2 is modulated by the adjacent negative regulatory segment.

DNA binding of heat shock factor 2 (HSF2) is induced during hemin-induced differentiation of human erythroleukemia cell line K562. To identify the transcriptional activation and the regulatory domains of HSF2, we constructed a series of deletion derivatives fused to the yeast GAL4 DNA binding domain and analyzed their transactivation activity. A minimal transactivation domain of HSF2 was localized to the C-terminus (residues 472-536), as in HSF1, although amino acid sequence similarity for these regions was rather limited and the potential transactivation ability was about 25% that of HSF1. The transactivation mediated by this region of HSF2 was found to be negatively regulated by the adjacent 18 amino acid segment (residues 428-445) under normal conditions. Furthermore, the latter segment, when fused to the GAL4 activation domain, markedly inhibited GAL4 activity. Extract containing most derivatives of HSF2 retaining this segment exhibited doublet or triplet bands in gel mobility shift assays with heat shock element-containing DNA, suggesting possible involvement of some factors interacting with that segment in the negative regulation. Another putative transactivation domain and two negative regulatory regions were also localized within the internal region.

Amino Acid Sequence↗

PubMed1988

A cAMP-dependent regulatory protein for RLC-a myosin kinase catalyzing the phosphorylation of scallop smooth muscle myosin light chain.

A cAMP-dependent regulatory protein which modulates the phosphorylation of scallop myosin regulatory light chain-a (RLC-a) by RLC-a myosin kinase (aMK) (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163) was purified from the scallop smooth muscle. RLC-a is abundant in the opaque portion of scallop smooth muscle, one of the catch muscles. The regulatory protein for aMK was purified by employing successively DEAE Toyopearl ion exchange chromatography, Sepharose 4B-8(6-aminohexylamino)cAMP affinity chromatography, and Sephadex G 100 gel filtration. The molecular mass of the regulatory protein was 41 kDa, based on the mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. With increasing amounts of the regulatory protein, the aMK activity decreased, and complete inhibition was observed at the concentration of twice that of aMK. The aMK activity inhibited by the regulatory protein was restored by the addition of cAMP. These results suggest that aMK is similar to a catalytic subunit of cAMP-dependent protein kinase, and the protein reported here is similar to its regulatory subunit. aMK may exist as an inactive form, as a combination with this regulatory protein, in vivo and be deinhibited by an increase in the intracellular concentration of cAMP. We discuss a possible correlation between the phosphorylation of RLC-a in myosin catalyzed by aMK and the catch state of the opaque portion of scallop smooth muscle.

Animals↗