Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Raf kinases are regulators of cellular proliferation, transformation, differentiation, and apoptosis. To identify downstream targets of Raf-1 in vivo, we used NIH 3T3 fibroblasts expressing a Raf-1 kinase domain-estrogen receptor fusion protein (BXB-ER), whose activity can be acutely regulated by estrogen. Proteins differentially phosphorylated 20 min after BXB-ER activation in living cells were displayed by two-dimensional electrophoresis. The protein with the most prominent newly induced phosphorylation was identified as stathmin, a phosphorylation-sensitive regulator of microtubule dynamics. Stathmin is rapidly phosphorylated on two ERK phosphorylation sites (serines 25 and 38) upon BXB-ER activation. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase (MEK) inhibitor PD98059 abolished this phosphorylation, demonstrating that stathmin is targeted by BXB-ER via the MEK/ERK pathway. Prolonged BXB-ER activation resulted in the accumulation of a stathmin phosphoisomer with impaired microtubule-destabilizing activity. The appearance of this phosphoisomer after BXB-ER activation correlated with rearrangements in the microtubule network, resulting in the formation of long bundled microtubules extending toward the rim of the cells. Our results identify stathmin as a main target of the Raf/MEK/ERK kinase cascade in vivo and strongly suggest that ERK-mediated stathmin phosphorylation plays an important role for the microtubule reorganization induced by acute activation of Raf-1.
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PMID:Activated raf induces the hyperphosphorylation of stathmin and the reorganization of the microtubule network. 971 20

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.
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PMID:Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol. 971 46

The pineal hormone, melatonin, inhibits proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells, modulates both ER mRNA and protein expression, and appears to be serum dependent, indicating interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed by transiently transfecting MCF-7 cells with an ERE-luciferase reporter construct. MCF-7 cells pre-treated with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen-independent transactivation of the ER. None of the compounds when used alone transactivated the ER. The ability of melatonin and EGF to transactivate the ER was abolished by the addition of the antiestrogen, ICI 164384, suggesting that melatonin and EGF co-operate to transactivate the ER. The modulation of ER transactivation was associated with changes in mitogen activated protein kinase activity and ER phosphorylation. This ER transactivation was blocked by pertussis toxin, a Galpha i-protein-coupled receptor inhibitor, suggesting cross talk between the G-protein-coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways in modulating ER transactivation. Exactly how the ability of melatonin in combination with EGF to transactivate the ER relates to melatonin's observed growth suppressive effects is not clear. It is possible that, although melatonin and EGF transactivate the ER, this transactivation does not result in the full transcription of estrogen-responsive genes, but rather, makes the ER refractory to activation by estradiol, thus, blocking the mitogenic actions of estradiol.
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PMID:Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors. 972 86

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.
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PMID:c-Myc is a major mediator of the synergistic growth inhibitory effects of retinoic acid and interferon in breast cancer cells. 980 32

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (delta) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as deltaA-Raf:ER was 20- to 200-fold more efficient than either deltaRaf-1:ER or deltaB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive deltaRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, deltaRaf:ER-infected cells. Abrogation of cytokine dependency by the activated deltaRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated deltaRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.
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PMID:Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. 984 21

Phosphorylation provides an important mechanism by which transcription factor activity is regulated. Estrogen receptor alpha (ERalpha) is phosphorylated on multiple sites, and stimulation of a number of growth factor receptors and/or protein kinases leads to ligand-independent and/or synergistic increase in transcriptional activation by ERalpha in the presence of estrogen. Here we show that ERalpha is phosphorylated by protein kinase A (PKA) on serine-236 within the DNA binding domain. Mutation of serine-236 to glutamic acid prevents DNA binding by inhibiting dimerization by ERalpha, whereas mutation to alanine has little effect on DNA binding or dimerization. Furthermore, PKA overexpression or activation of endogenous PKA inhibits dimerization in the absence of ligand. This inhibition is overcome by the addition of 17beta-estradiol or the partial agonist 4-hydroxy tamoxifen. Interestingly, treatment with the complete antagonist ICI 182,780 does not overcome the inhibitory effect of PKA activation. Our results indicate that in the absence of ligand ERalpha forms dimers through interaction between DNA binding domains and that dimerization mediated by the ligand binding domain only occurs upon ligand binding but that the complete antagonist ICI 182,780 prevents dimerization through the ligand-binding domain. Heterodimer formation between ERalpha and ERbeta is similarly affected by PKA phosphorylation of serine 236 of ERalpha. However, 4-hydroxytamoxifen is unable to overcome inhibition of dimerization by PKA. Thus, phosphorylation of ERalpha in the DNA binding domain provides a mechanism by which dimerization and thereby DNA binding by the estrogen receptor is regulated.
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PMID:Phosphorylation of human estrogen receptor alpha by protein kinase A regulates dimerization. 989 Oct 36

17Beta-estradiol can potentiate kainate-induced currents in isolated hippocampal CA1 neurons. The action of estrogen was rapid in onset, steroid and stereospecific, and reversible. The potentiation could be mimicked by 8-bromo-cAMP, an activator of protein kinase A. As the hippocampus expresses both isoforms of the intracellular estrogen receptor (ER alpha and ER beta), the role of ERs in the rapid action of 17beta-estradiol remains elusive. Here we report that the rapid action of 17beta-estradiol is independent from the classical ER activation in the modulation of membrane excitability. Under whole cell voltage clamp recording configuration, 17beta-estradiol-induced potentiation was observed in both wild-type and the ER alpha gene knockout mice. The perfusion or incubation of ICI 182,780, which blocks both ER alpha and ER beta, did not affect estrogen potentiation in either group. Further study showed that adenosine 3',5'-cyclic-monophosphothioate Rp-isomer, a specific inhibitor of protein kinase A, completely blocked the potentiation observed with the application of 17beta-estradiol in ER alpha gene knockout mice. Our results provide evidence that a distinct estrogen-binding site exists, which appears to be coupled to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid/kainate receptors by a cAMP-dependent phosphorylation process.
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PMID:Rapid action of 17beta-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors. 992 91

We have shown that estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing CNS. We subsequently demonstrated widespread colocalization of estrogen and neurotrophin receptors (trk) within developing forebrain neurons and reciprocal transcriptional regulation of these receptors by their ligands. Using organotypic explants of the cerebral cortex, we tested the hypothesis that estrogen/neurotrophin receptor coexpression also may result in convergence or cross-coupling of their signaling pathways. Estradiol elicited rapid (within 5-15 min) tyrosine phosphorylation/activation of the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, that persisted for at least 2 hr. This extracellular signal-regulated protein kinase (ERK) activation was inhibited successfully by the MEK1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780, and did not appear to result from estradiol-induced activation of trk. Furthermore, we also found that estradiol elicited an increase in B-Raf kinase activity. The latter and subsequent downstream events leading to ERK activation may be a consequence of our documentation of a multimeric complex consisting of, at least, the ER, hsp90, and B-Raf. These novel findings provide an alternative mechanism for some of the estrogen actions in the developing CNS and could explain not only some of the very rapid effects of estrogen but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation.
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PMID:Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. 995 96

In an attempt to find the key to reducing the excessive morbidity and mortality seen with mood disorders, our laboratory has been extensively investigating lithium's mechanisms of action in an integrated series of clinical and preclinical studies. We have found that the chronic administration of the 2 structurally highly dissimilar agents, lithium and valproate, brings about a strikingly similar reduction in protein kinase C (PKC) alpha and epsilon isozymes in rat frontal cortex and hippocampus. In view of PKC's critical role in regulating neuronal excitability and neurotransmitter release, we have postulated that PKC inhibition may have antimanic efficacy. In a small study, we have found that tamoxifen (which, in addition to its estrogen receptor blockade, is also a PKC inhibitor) has marked antimanic efficacy. These exciting preliminary results suggest that PKC inhibitors may represent a novel class of improved therapeutic agents for bipolar disorder, and this is under further investigation. The beneficial effects of mood stabilizers require a lag period for onset of action and are generally not immediately reversed upon drug discontinuation; such patterns of effects suggest alterations at the genomic level. We have therefore undertaken a series of studies to investigate the effects of these agents on the AP-1 family of transcription factors and have found that both drugs increase AP-1 DNA binding activity in areas of rodent brain ex vivo and in human neuronal cells in culture. Both treatments also increase the expression of a reporter gene driven by an AP-1-containing promoter, and mutations in the AP-1 sites of the reporter gene promoter markedly attenuate these effects. Both treatments also increase the expression of several endogenous proteins, whose genes are known to be regulated by AP-1. Although the precise mechanisms have not been fully elucidated, preliminary results suggest that these effects may be mediated, in part, by mitogen-activating protein kinases and glycogen synthase kinase 3beta. We have also utilized mRNA reverse transcription-polymerase chain reaction (RT-PCR) differential display to identify concordant changes in gene expression induced by the chronic administration of both lithium and valproate. We have identified concordant changes in a number of cDNA bands by both lithium and valproate. Cloning and characterizing of these genes is currently underway. The identification of the functions of these genes offers the potential not only for improved therapeutics for reducing the morbidity and mortality associated with mood disorders, but may also provide important clues about the underlying pathophysiology.
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PMID:Modulation of CNS signal transduction pathways and gene expression by mood-stabilizing agents: therapeutic implications. 1007 85

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.
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PMID:Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. 1009 55


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