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)

The regulation of S6 kinase activity was used to monitor perturbations of intracellular signaling activity during heat shock of quiescent murine and human fibroblasts. Previous reports on exponentially growing insect and plant cells had indicated that 40S ribosomal protein S6 is dephosphorylated during heat shock; thus inhibition of S6 kinase activity by heat shock was anticipated in NIH 3T3 fibroblasts and human cells (HeLa, diploid embryonic fibroblasts MRC-5, and skin-derived fibroblasts). Unexpectedly, two distinct S6 protein kinases were activated in quiescent fibroblasts after heat exposure. One of the enzymes was partially purified by sequential column chromatography and was determined to be equivalent to the enzyme activated by serum and other growth factors, referred to here as pp70-S6 protein kinase. The other protein S6 kinase, pp90rsk, was identified by a specific immunoprecipitation assay. Monitoring both enzymatic activities during heat shock revealed a temporal pattern of activation that was reversed when compared to non-stressed, mitogen-stimulated cells. Finally, heat shock stimulated protein S6 phosphorylation in cultured, quiescent mammalian cells. These data demonstrate that specific protein kinases can be activated during heat shock, and that some early mitogenic signals may also participate in the response of cells to physiologic stress.
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PMID:Heat shock induces two distinct S6 protein kinase activities in quiescent mammalian fibroblasts. 188 Jan 53

Prostaglandins (PGs) are multi-potent mediators for local tissue homeostasis and inflammatory reactions. Addition of PGs to cultures of human skin fibroblasts led to a marked induction of the production of hepatocyte growth factor (HGF). PGE1 and PGI2 analogues were the most potent in stimulating HGF production by over 50-fold; PGE2 and PGD2 were less potent. Western immunoblotting of conditioned medium from skin fibroblasts indicated that both PGE1, PGE2, and PGI2 analogues specifically induce synthesis of HGF with a M(r) of 85,000, but not smaller variant of HGF with a M(r) of 28,000. Consistent with the induction of HGF production, steady-state expression of HGF mRNA in fibroblasts was strongly induced by PGE1 and PGI2 analogues, but slightly by PGE2 and PGD2, thereby indicating that PGs induce HGF production through transcriptional activation of the HGF gene. PGE1, PGE2, PGI2 analogue also stimulated HGF production in MRC-5 human embryonic lung fibroblasts and vascular smooth muscle cells. As dibutyryl cyclicAMP (dbcAMP) and tetradecanoylphorbol 13-acetate (TPA), but not Ca(2+)-ionophore A23187 stimulated HGF production in skin fibroblasts, activation of protein kinase-A and protein kinase-C may be coupled to the stimulation of HGF production. Simultaneous addition of PGE1 and TPA or dbcAMP and TPA led to a synergistic enhancement of HGF production, whereas the simultaneous addition of PGE1 and dbcAMP failed to additively enhance HGF production.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Novel function of prostaglandins as inducers of gene expression of HGF and putative mediators of tissue regeneration. 760 39

Two-dimensional (2-D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2-D gels, the analysis of phosphoproteins, can provide much information as to the assembly and "wiring" of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P-labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P- and 32P-labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2-D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and microsequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2-D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin-1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat-shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC-5 human lung fibroblasts and their SV-40-transformed counterparts, MRC-5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2-D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.
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PMID:Analysis of cellular phosphoproteins by two-dimensional gel electrophoresis: applications for cell signaling in normal and cancer cells. 805 70

We have recently isolated the cDNA for the murine homologue of the stress-inducible phosphoprotein STI1 (also known as IEF SSP 3521 or p60). STI1 was previously shown to be 2-fold up-regulated in MRC-5 fibroblasts upon viral transformation and to exist in a macromolecular complex with heat shock proteins of the HSP 70 and 90 families. By peptide-sequencing we have identified the two heat shock proteins that bind to murine STI1 (mSTI1) as HSC 70 and HSP 84/86. We describe two separate binding regions within mSTI1 for the two heat shock proteins. In the presence of cell extracts, the N-terminal region of mSTI1 binds preferentially to HSC 70, whereas the C-terminal portion of the molecule promotes the binding of HSP 84/86. Heat treatment caused a strong induction of mSTI1 message without affecting the steady-state level of the protein significantly. In addition, heat treatment led to changes in the isoform-composition of mSTI1. pp70(s6k), pp90(rsk), and mitogen-activated protein kinase-activated protein kinase 2 were tested as possible STI1 kinases in vitro using recombinant mSTI1 as a substrate: only pp90(rsk) was able to phosphorylate recombinant mSTI1. In vitro kinase assays using casein kinase II suggest serine 189 to be a likely phosphorylation site in mSTI1.
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PMID:Stress-inducible, murine protein mSTI1. Characterization of binding domains for heat shock proteins and in vitro phosphorylation by different kinases. 899 75

In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa
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PMID:Expression, purification, and characterization of DNA polymerases involved in papovavirus replication. 902 36

In the previous paper (Ookata et al., (1997) Biochemistry, 36: 249-259), we identified two mitotic cdc2 kinase phosphorylation sites (Ser696 and Ser787) in the proline-rich region of human MAP4. One (Ser696) of them was also phosphorylated during interphase. A protein kinase responsible for interphase phosphorylation of Ser696 could necessarily be distinct from cdc2/cyclin B kinase. To get insights into a physiological role for Ser696 phosphorylation, we searched for a Ser696 kinase and for cellular conditions under which Ser696 is dephosphorylated. Because Ser696 conforms to the MAP kinase phosphorylation consensus motif (PXSP), MAP kinase was tested as a possible kinase phosphorylating Ser696. MAP kinase, in fact, did phosphorylate Ser696 in MTB3, the carboxy-terminal half of human MAP4 in vitro. Phosphorylation of Ser696 in HeLa cell extract was suppressed by a MAP kinase inhibitor, DBTM-0004. Also consistent with the notion that Ser696 is a MAP kinase site were the fact that serum-starvation induced dephosphorylation of Ser696 in HeLa cells, TIG-3 and MRC-5-30 human fibroblasts, while readdition of serum recovered Ser696 phosphorylation, albeit after a surprisingly long interval. Thus, phosphorylation of Ser696 of MAP4, most likely carried out by MAP kinase, may play a role in modulation of MAP4 activity in proliferating versus quiescent cells.
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PMID:Serum-dependent phosphorylation of human MAP4 at Ser696 in cultured mammalian cells. 1521 89

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.
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PMID:Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma. 1554 42

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing alpha-smooth muscle actin (alpha-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of alpha-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell-cell contact or serum starvation, and examined the relationship between decorin and alpha-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell-cell contact. In contrast, the expression of alpha-SMA in cells made quiescent by cell-cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of alpha-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of alpha-SMA.
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PMID:Effects of decorin on the expression of alpha-smooth muscle actin in a human myofibroblast cell line. 1795 60

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.
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PMID:Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells. 1849 64

Cancer cells appear to depend heavily on antiapoptotic proteins for survival and so targeted inhibition of these proteins has therapeutic potential. One innovative strategy is to inhibit the cyclin-dependent kinases (CDKs) responsible for the regulation of RNA polymerase II (RNAPII). In our study, we investigated the detailed cellular mechanism of a novel small-molecule CDK inhibitor (CDKI-71) in cancer cell lines, primary leukemia cells, normal B - & T- cells, and embryonic lung fibroblasts and compared the cellular and molecular responses to the clinical CDK inhibitor, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis induction that were closely associated with the inhibition of RNAPII phosphorylation at serine-2. This was caused by effective targeting of cyclinT-CDK9 and resulted in the downstream inhibition of Mcl-1. No correlation between apoptosis and inhibition of cell-cycle CDKs 1 and 2 was observed. CDKI-71 showed a 10-fold increase in potency in tumor cell lines when compared to MRC-5 human fibroblast cells. Significantly, CDKI-71 also demonstrated potent anti-chronic lymphocytic leukemia activity with minimal toxicity in normal B- and T-cells. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Here, we provide the first cell-based evidence that flavopiridol induces DNA double-strand breaks: a fact which may explain why flavopiridol has such a narrow therapeutic window in preclinical and clinical settings. Taken together, our data provide a rationale for the development of selective CDK inhibitors as therapeutic agents and CDKI-71 represents a promising lead in this context.
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PMID:CDKI-71, a novel CDK9 inhibitor, is preferentially cytotoxic to cancer cells compared to flavopiridol. 2148 92


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