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PubMed1986

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase.

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M(2) seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH(2)-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c(1)NR), constitutive NADH:NR (c(2)NR), and nitrate-inducible NR (iNR).

Journal Article↗
PubMed2001

The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens.

Jasmonates (JAs) inhibit plant growth and induce plant defense responses. To define genes in the Arabidopsis JA signal pathway, we screened for mutants with constitutive expression of a luciferase reporter for the JA-responsive promoter from the vegetative storage protein gene VSP1. One mutant, named constitutive expression of VSP1 (cev1), produced plants that were smaller than wild type, had stunted roots with long root hairs, accumulated anthocyanin, had constitutive expression of the defense-related genes VSP1, VSP2, Thi2.1, PDF1.2, and CHI-B, and had enhanced resistance to powdery mildew diseases. Genetic evidence indicated that the cev1 phenotype required both COI1, an essential component of the JA signal pathway, and ETR1, which encodes the ethylene receptor. We conclude that cev1 stimulates both the JA and the ethylene signal pathways and that CEV1 regulates an early step in an Arabidopsis defense pathway.

Arabidopsis↗
PubMed2001

Constitutional values and HIV/AIDS in the workplace: reflections on Hoffman v. South African Airways.

South Africa is experiencing an HIV/AIDS epidemic of enormous proportions. The workplace, like all the other sectors, is adversely affected. The tendency of a significant proportion of employers has been to discriminate against employees and job applicants living with HIV/AIDS through the use of HIV testing to exclude those that are HIV-positive. In the case of Hoffman v South African Airways, the Constitutional Court was asked to determine the constitutionality of excluding a job applicant on account of an HIV-positive status. The Court ruled in favour of the applicant and ordered instatement. Through an analysis of Hoffman v South African Airways, the article examines how constitutional values are impacting on HIV/AIDS-related ethics in the workplace.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Constitutive expression of both subunits of interleukin-12 in human keratinocytes.

Interleukin-12 (IL-12), a heterodimeric molecule consisting of disulfide-linked 35- and 40-kDa chains, is secreted by a variety of cells including macrophages and B cells. While keratinocytes have recently been demonstrated to produce IL-12 after stimulation with phorbol-12,13-dibutyrate as well as trinitrochlorobenzene, the constitutive expression of the two IL-12 subunits has remained controversial. In this study, we investigate if cultured keratinocytes derived from human epidermis and the follicle outer root sheath constitutively express IL-12. Total RNA was reverse transcribed to cDNA and amplified using a highly sensitive nested reverse transcriptase polymerase chain reaction. Both IL-12 p40 and p35 transcripts were detected in keratinocyte cultures. Moreover, low levels of the IL-12 p70 heterodimer were detected in the culture supernatants, as determined by a sensitive enzyme-linked immunosorbent assay. Since IL-12 is known to play an important role in the development of Th1 cells, the constitutive expression of mRNA for IL-12 in keratinocytes together with its secretion adds further evidence for a role of keratinocytes in immunological processes within the skin such as in contact hypersensitivity.

Base Sequence↗
PubMed1993

Human keratinocytes in vivo and in vitro constitutively express the 72-kD heat shock protein.

Exposure of cells to elevated temperatures induces a physiologic response characterized by the synthesis of a specific set of proteins (heat shock or stress proteins, HSPs) mediating repair mechanisms and protection from cellular damage. In the present study upon immunohistochemistry using a specific monoclonal antibody, the constitutive and heat-induced expression of the 72-kD HSP (HSP72) in normal human skin and in human epidermal cell lines (KB, A431) was investigated. Normal (unstressed) epidermis and adnexal structures of normal human skin were found to constitutively express HSP72. In contrast, a substantial HSP72 expression could not be observed in the dermal cellular compartment. In vitro heat treatment of punch biopsies from normal skin (42 degrees C, 4 h) resulted in a further increase of epidermal HSP72 expression. In addition, dermal cells were found to be induced to express HSP72. To further evaluate the spontaneous HSP72 expression of epidermal cells two epidermoid carcinoma cell lines (A431, KB) were investigated. Upon immunohistochemistry and Western blot analysis a significant HSP72 expression could be detected in unstressed KB and A431 cells. In contrast, a human fibrosarcoma cell line (HT1080) was negative for HSP72 at 37 degrees C but upon heat treatment a strong induction was observed. Furthermore, Northern blot analysis using a cDNA probe specific for human HSP72 revealed a constitutive expression of HSP72 mRNA in both epidermal cell lines. These findings demonstrate a significant expression of the stress-inducible HSP72 in unstressed human skin as well as in epidermal cell lines, suggesting that HSP72 may inherently be involved in the protective function of normal human skin.

Blotting, Northern↗
PubMed2002

Differential mechanisms of constitutive Akt/PKB activation and its influence on gene expression in pancreatic cancer cells.

Activated Akt/protein kinase B transmits oncogenic signals leading to inhibition of apoptosis, cellular proliferation, and tolerance to hypoxia. Presently, mutational inactivation of PTEN and activation of Ras are considered to be the major causes of Akt activation. Here we report differential mechanisms of constitutive Akt activation in 4 human pancreatic cancer cell lines (KMP-3, KMP-4, PCI-66, and PCI-68). These 4 cell lines displayed phosphorylation and functional activation of Akt both in the presence and absence of serum, while three control cell lines (PCI-79, KMP-8, and PSN-1) did so only in the presence of serum in culture. All the 7 cell lines harbored K-Ras activated by mutations at codon 12 resulting in MAP kinase kinase (MEK1/2) phosphorylation, and all except one (KMP-8) had p53 mutations, indicating that these mutations are not sufficient for constitutive Akt activation. KMP-3 and KMP-4 had lost PTEN function owing to loss of expression or a mutation, but PCI-66 and PCI-68 retained wild-type PTEN. Phosphorylation of Akt was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and the tyrosine kinase inhibitor genistein in KMP-3 and KMP-4 cells, indicating that upstream signals are required for Akt activation in these two cell lines. In contrast, neither LY294002 nor genistein inhibited Akt activation in PCI-66 and PCI-68 cells, indicating the involvement of another unknown mechanism of Akt activation independent of PI3K-mediated signaling to Akt. Irrespective of the differential mechanisms, the 4 cell lines showed similar mRNA expression patterns of 49 genes assessed by cDNA array as compared to the 3 cell lines without Akt activation, suggesting that the mechanisms have the same consequences on the downstream signaling of the constitutive Akt activation.

Chromones↗
PubMed2003

Cyclooxygenase-2 inhibitor NS-398 suppresses cell growth and constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells.

We previously established two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta stimulated the expression of G-CSF, GM-CSF, and cyclooxygenase (COX)-2 in the two cell lines. It is known that increased COX-2 activity promotes tumor growth and induces G-CSF and GM-CSF expression in non-malignant cells, and that selective COX-2 inhibitors inhibit the growth of some types of malignant cells. Therefore, we hypothesized that inhibition of COX-2 activity might suppress constitutive production of G-CSF or GM-CSF in addition to reducing the growth of malignant cells. We confirmed that the selective COX-2 inhibitor, NS-398 suppressed the constitutive production of G-CSF and GM-CSF, and the cell growth in both OKa-C-1 and MI-4 cell lines. Prostaglandin E2 (PGE2) reversed the inhibitions of G-CSF and GM-CSF expression, as well as cell growth, by NS-398. This result confirms that the effects of NS-398 are based on the inhibition of COX activity. Some studies have indicated that nuclear factor kappa B (NF-kappaB) or MAPK (mitogen-activated protein kinase) activation is related to upregulation of G-CSF, GM-CSF or COX-2 expression in some types of cells. Therefore, we examined if the actions of NS-398 might be mediated by the MAP kinase pathway or NF-kappaB activity in OKa-C-1 and MI-4 cells. We found that NS-398 inhibits G-CSF and GM-CSF production and cell growth through an extracellular signal-regulated kinase kinase (MEK) signaling pathway in these cell lines. The prognosis of non-small cell lung cancer showing G-CSF gene expression is significantly worse. G-CSF overproduction by tumor cells is observed at an advanced clinical stage. Our findings imply that a COX-2 inhibitor might improve the prognosis of patients with lung cancer through the reduction of G-CSF or GM-CSF.

Cell Division↗
PubMed1993

Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium.

Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two-component regulatory system. The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence-specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription. In this report, we describe a VirG mutant which is able to activate vir gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides. A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested. A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype. In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule. The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype. However, the vir genes were not induced to high levels when compared with the levels of the constitutive virG of strain A348.

Agrobacterium tumefaciens↗
PubMed2005

A constitutively active GPCR retains its G protein specificity and the ability to form dimers.

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.

Amino Acid Sequence↗
PubMed1992

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.

Animals↗
PubMed2006

Hybrid space: constituting the hospital as a home space for patients.

A growing body of nursing writing is engaged in reviewing the material and relational world of nursing using geographical concepts. This paper draws upon research undertaken in hospital settings where nurses constituted the hospital as a home space for patients. Nurses' practices created an equitable and patient-centred use of physical space in the hospital ward, along with the intimate, extended and personal relationships associated by patients with a caring and homely environment. It is suggested that this constitution of space resonates with the geographic notion of the therapeutic landscape and can be read as a challenge to more conventional uses of hospital spaces shaped by biomedical concerns. The implications of merging the distinctly different spaces and places of home and hospital are also explored using the concepts of hybridity and spatial vulnerability. The complex hybrid nursing creation of home space within hospital places works against the grain of usual understandings of hospitals as specialised illness spaces that are the domain of the health professional inhabitants. While there are limitations and dangers in the representation, this constitution of space is an expression of core nursing values privileging patient-nurse relationships and supports the maintenance of identity and personal expression.

Attitude of Health Personnel↗
PubMed1986

Different break-points in Philadelphia chromosome variant translocations and in constitutional and sporadic translocations.

Three different samples of translocations were considered in an attempt to identify those chromosomal bands preferentially involved in variant Philadelphia chromosome (Ph1) translocations, and to compare them with bands preferentially broken in constitutional and sporadic translocations. The first sample included 204 cases of variant Ph1 translocations with 221 identified break-points (bp), the second consisted of 106 cases of non-Robertsonian constitutional translocations with 213 bp identified, and the third one of 185 bp identified in sporadic translocations found occasionally in single cells of subjects with normal karyotypes and without haematological disorders. A statistical analysis demonstrated that there are some bands preferentially broken in each of the samples, and this with a high level of significance (P less than 0.001). The analysis of the distributions of the chi 2 components permitted us to identify the 12 bands preferentially involved in variant Ph1 translocations and the 13 and 9 bands preferentially involved in constitutional and sporadic translocations, respectively. The comparison among the groups of preferential bp showed that the bands most involved in the three samples are different. Some theoretical problems related to the origin of the Ph1 chromosome are discussed.

Chromosome Banding↗
PubMed2005

Constitutive high-affinity choline transporter endocytosis is determined by a carboxyl-terminal tail dileucine motif.

Maintenance of acetylcholine synthesis depends on the effective functioning of a high-affinity sodium-dependent choline transporter (CHT1). Recent studies have shown that this transporter is predominantly localized inside the cell, unlike other neurotransmitter transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylcholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis.

Amino Acid Motifs↗
PubMed2006

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors.

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.

Analgesics, Opioid↗
PubMed1996

Constitutive cytokine release by simian T-cell lymphotrophic virus type I (STLV-I) and human T-cell lymphotrophic virus types I/II (HTLV-I/II) transformed cell lines.

Simian T-cell lymphotrophic virus type I (STLV-I) and human T-cell lymphotrophic virus types I/II (HTLV-I/II) contain the tax gene which can transactivate the transcription of viral and cellular genes including several cytokines. These investigations used two STLV-I and four different HTLV-I/II transformed cell lines to quantitate constitutive cytokine release, p24 antigen production, and to correlate gag p24 antigen and cytokine release. These investigations are the first to report 1) quantitative comparison of constitutive release of multiple cytokines by several STLV-I and HTLV-I/II transformed cell lines and to determine the cytokines constitutively released by STLV-I transformed cell lines as IL-6, b-FGF, and GM-CSF (and TNF-beta, and PDGF in a higher viral producing line); 2) statistically significant differences in levels of cytokines produced by STLV-I and HTLV-I/II transformed cell lines, dependent on the method of results quantification; and 3) a correlation between levels of STLV-I and HTLV-I/II and gag p24 antigen production and cytokine production.

Animals↗
PubMed1999

Relationship between constitutive skin color and ultraviolet light sensitivity in Koreans.

The assessment of skin sensitivity to ultraviolet (UV) radiation is important in treating a variety of skin diseases and preventing the deleterious effects of UV. Although there are many ways to predict the sensitivity to UVR, controversies exist over their objectivity, correlation, and applicability in various races. For the purpose of evaluating the relationship of skin color and UV responsiveness, this study was performed to assess the validity of constitutive skin color for an individual's UV sensitivity in 80 healthy Korean volunteers. The subjects were divided into two groups: a young-aged group with 40 subjects aged 20-39 years and an old-aged group with 40 subjects aged 43-63 years. A minimal erythema dose (MED) of UVB and minimal immediate pigment darkening dose (MIPDD) of UVA were assessed using a fluorescent lamp (Panasol II, National Biologic Co., Twinberg, USA) and a metal-halide lamp (Dermalight 2001, Mutzhas, Munich, Germany), respectively. The constitutive skin color of buttock was measured with a Minolta Chroma Meter CR-300 (Osaka, Japan), using the CIE (Commission International de l'Eclairege) L*a*b* system. Of the three L*a*b*, we used the L* value to estimate the skin color. For the MED and L* values, the values were inversely proportionate (P<0.05) in the young-aged group; however, no correlation existed in the old-aged group. No relationships existed among Fitzpatrick skin type and MED, skin type and MIPDD. The results of this study indicated that the constitutive skin colors in the young-aged group can be an objective and adequate predictor of cutaneous UV sensitivity.

Adult↗
PubMed2004

Inter-relation between variables determining constitutional UV sensitivity in Caucasian children.

BACKGROUND/PURPOSE: Sensitivity to ultraviolet (UV) light is associated with phenotypic variables like iris or hair colour or freckling, which are all strongly inter-related. We assessed the pattern of association among these factors to construct a summary measure reflecting constitutional UV sensitivity in a simple one-dimensional score with sufficient discriminating power. METHODS: Information on iris and hair colour (3 and 4 categories, respectively), freckling (none vs. any) and the reaction to mid-day summer sunlight as an outcome (dichotomized into 'burn' and 'tan') was collected in a cross-sectional study among children at school enrollment in 1999 and 2000 in Göttingen, Germany (n=3765 with complete data). Chi-squared automatic interaction detector (CHAID) analysis and log-linear modelling was used to assess the pattern of mutual inter-relations. RESULTS: Hair colour turned out to be the factor most strongly associated with UV sensitivity. In case of blonde or brown hair (the most common phenotypes in our sample), the consideration of freckling further refined the classification system, whereas iris colour did not contribute additional information. The results of the CHAID and log-linear analyses suggest a simple five-point score capturing constitutional UV sensitivity. The discriminating power of this score was high in our sample, with the proportion of persons who burned ranging from 7.7 (lowest category) to 73% (highest category). CONCLUSION: The new scoring system summarized efficiently relevant information on individual constitutional UV sensitivity in our sample. Its further validation in independent studies comprising Caucasian children or adults is necessary to assess objectively its properties for future applications.

Child↗
PubMed2006

Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade.

Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.

AMP-Activated Protein Kinases↗