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Query: UNIPROT:P06889 (Mol)
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The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506-541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Biol Rep 1993 Jun
PMID:Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens. 769 76

Previously we reported that mutations in the Saccharomyces cerevisiae REG1 gene encoding a negative regulator of glucose-repressible genes, suppress the RNA processing defects and temperature-sensitive growth of rna1-1 and prp cells. This result and the fact that growth on non-glucose carbon sources also suppresses rna1-1 led us to propose that RNA processing and export of RNA from the nucleus are responsive to carbon source regulation. To understand how carbon source affects these processes, we used p70, an antigen regulated by REG1 and by glucose availability, as a reporter. We found that the response of p70 to glucose availability is mediated by both the SNF1-SSN6-dependent glucose repression and the RAS-cAMP pathways. These results led us to test whether the RAS-cAMP pathway interacts with RNA1. We found that suppression of rna1-1 appears to be mediated, at least in part, by the RAS-cAMP pathway.
Mol Gen Genet 1995 Apr 10
PMID:The glucose repression and RAS-cAMP signal transduction pathways of Saccharomyces cerevisiae each affect RNA processing and the synthesis of a reporter protein. 771 3

Previous studies have shown that the noncatalytic carboxy-terminal tail of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibitory pseudosubstrate domain that is phosphorylated in situ during activation and in vitro by mitogen-activated protein kinases. The present study shows that a recombinant p70 deleted of the carboxy-terminal tail (p70 delta CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin very similar to those of the parent, full-length p70 kinase. Carboxy-terminal deletion reduces the extent of maximal inhibition produced by rapamycin, from > 95% in the full-length p70 to 60 to 80% in p70 delta CT104, without altering the sensitivity to rapamycin inhibition (50% inhibitory concentration of 2 nM). Serum activation of p70 delta CT104, as with the parent, full-length p70, is accompanied by an increase in 32P content (about twofold) in situ and a slowing in electrophoretic mobility; both modifications are inhibited by pretreatment with wortmannin or rapamycin. 32P-peptide maps of p70 delta CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferential dephosphorylation of the same subset of sites. Thus, it is likely that activation of the kinase requires phosphorylation of p70 at sites in addition to those previously identified in the carboxy-terminal tail. Evidence that the carboxy-terminal tail actually functions as a potent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region. Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continue phosphorylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70 delta 29-46/ delta CT104) increases activity 10-fold, to a level approaching that of p70 delta CT104. Deletion of residues 29 to 46 also abolishes completely the sensitivity of p70 to inhibition by rapamycin but does not alter the susceptibility to activation by serum of inhibition by wortmannin. Although the mechanisms underlying the effects of the delta 29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phosphorylation site at Ser-40. Thus, activation of the p70 S6 kinase involves multiple, independent inputs directed at different domains of the p70 polypeptide. Disinhibition from the carboxy-terminal tail requires, in addition to its multisite phosphorylation, an activating input dependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct from that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase. In addition, as exemplified by the rapamycin-resistant but mitogen- and wortmannin-sensitive p70 delta 29-46/ delta CT104 mutant, a further activating input, which probably involves site-specific phosphorylation in the segment between amino acids 46 to 421, is necessary.
Mol Cell Biol 1995 May
PMID:Multiple independent inputs are required for activation of the p70 S6 kinase. 773 16

Prothoracicotropic hormone (PTTH)-stimulated protein phosphorylation leads to ecdysteroidogenesis (molting hormone biosynthesis) in the prothoracic glands of the tobacco hornworm, Manduca sexta. The phosphorylation of 34 and 50 kDa peptides (p34 and p50) paralleled the increase in ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preceded a decrease in ecdysteroidogenesis. Inhibition by rapamycin of p34, but not p50, phosphorylation prevented PTTH-stimulated ecdysteroidogenesis in a dose-dependent manner, suggesting that p34 phosphorylation is requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whose synthesis was rapidly stimulated by PTTH were p50 and p70. The time-course of PTTH-stimulated synthesis of p50 paralleled that of p34 phosphorylation and that of ecdysteroidogenesis. Rapamycin inhibited PTTH-stimulated synthesis of p50 and p70, suggesting that specific protein synthesis is also required for PTTH-stimulated ecdysteroidogenesis, confirming the results of Rybczynski and Gilbert [(1994) Insect Biochem. Molec. Biol. 24, 175-189], and that p34 phosphorylation may regulate the downstream synthesis of p50 and p70, possible key regulatory proteins leading to ecdysteroidogenesis. Results from two-dimensional (2D)-PAGE analysis of the ribosomal proteins purified from prothoracic glands, demonstrated that p34 is indeed ribosomal S6, and is phosphorylated at up to five sites (P1-5) upon PTTH stimulation. The multiple phosphorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Temporal analysis of PTTH-stimulated S6 phosphorylation by 2D-PAGE revealed that phosphorylation of S6 at the P1 site was temporally correlated with the initiation of ecdysteroidogenesis, and that multiple phosphorylation at all five sites (P1-5) was correlated with the maximal synthesis of ecdysteroids. Dephosphorylation of S6 was accompanied by a decrease in ecdysteroidogenesis. These data demonstrate that p34 is ribosomal protein S6 and that both the phosphorylation of S6 and specific protein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland.
Insect Biochem Mol Biol 1995 May
PMID:Multiple phosphorylation of ribosomal protein S6 and specific protein synthesis are required for prothoracicotropic hormone-stimulated ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta. 778 42

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.
Mol Cell Biol 1994 Nov
PMID:Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. 793 49

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
Mol Cell Biol 1994 Dec
PMID:Replication factor A is required in vivo for DNA replication, repair, and recombination. 796 28

p56lck, a src family protein tyrosine kinase interacts with several T cell receptors, like: CD4, CD8, CD2 and the beta-chain of the IL2, thereby receptors devoid of kinase activity may transduce signals via tyr phosphorylation. Tyr 192 and ser 194, located in the SH2 domain of p56lck is phosphorylated upon CD3 triggering, which can change interactions of tyr-P proteins with this SH2 domain. Upon activation through the CD2 or the CD45 receptors the kinase activity of p56lck is temporarily increased. By immunofluorescent and confocal microscopy we observed that a significant proportion of p56lck and CD2 receptors are localized in endosomal vesicles after stimulation. By Western blot we showed a parallel recruitment of the PTK p70-ZAP in this vesicles. The role of p56lck away from the plasma membrane localized in vesicles is under study.
Cell Mol Biol (Noisy-le-grand) 1994 Jul
PMID:P56lck A lymphocyte specific protein tyrosine kinase: activation, regulation and signal transduction. 798 18

The prothoracic glands of fifth instar Manduca sexta larvae respond to stimulation by the brain neuropeptide, prothoracicotropic hormone (PTTH), with a several-fold increase in the rate of ecdysteroid synthesis. Previous studies have shown that this response requires protein synthesis and that the action of PTTH can be mimicked by dibutyryl cAMP (dbcAMP) and the Ca2+ ionophore, A23187. To further understand the role of protein synthesis in the response of prothoracic glands to PTTH, patterns of protein synthesis in stimulated glands were examined using glands incubated in vitro with [35S]methionine. All three agents caused an increase in the rate of ecdysteroid synthesis as well as an increase of up to 300% in the synthesis and/or accumulation of three proteins (p100, p70, and p"50") within 2 h of stimulation. Changes in these three proteins were specific to the prothoracic gland, were not elicited by non-brain peptides and were not simply a result of increased general protein synthesis in the gland. Exposure of the glands to A23187 alone, or concurrently with dbcAMP, resulted in increased synthesis of p100, p70, p"50" and ecdysteroids but decreased general protein synthesis. Increased synthesis of these proteins could be detected within 15 min after initiating PTTH stimulation. The behavior of these three proteins makes them candidates for modulators of ecdysteroid synthesis in the prothoracic gland. The results suggest also that PTTH may activate two biochemical pathways in the gland: one path leading to increased synthesis of the p100, p70, and p"50" proteins and increased ecdysteroid synthesis, and the second leading to increased general protein synthesis. This second trophic effect is vulnerable to intracellular Ca2+ changes that do not inhibit the first pathway.
Insect Biochem Mol Biol 1994 Feb
PMID:Changes in general and specific protein synthesis that accompany ecdysteroid synthesis in stimulated prothoracic glands of Manduca sexta. 811 26

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.
Mol Cell Biol 1994 Feb
PMID:Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction. 828 87

The mitogen response of p70/p85 S6 kinase (S6K) parallels that of mitogen-activated protein kinases (MAPK). However, S6K lies on a discrete signaling pathway from MAPK, since the immunosuppressant drug rapamycin inactivates S6K without affecting the MAPK cascade. Phosphatidylinositol 3-kinase operates upstream of S6K, but the intermediate effectors in this signaling pathway are unknown. We have identified an autoinhibitory domain in S6K that overrides the requirement of the amino terminus for the activation of S6K. The region between codons 58 and 77 is highly inhibitory, and its deletion results in constitutive kinase activation. Additionally, deletion of the first 77 codons confers mitogen independence and insensitivity to rapamycin. Rat1 cells expressing delta N77 S6K exhibit a distinctly abnormal morphology. This constitutively active mutant will provide a useful means of studying the effects of expressing unregulated S6K in cells. Subdeletion analysis of the amino terminus has defined two discrete domains in the N terminus of S6K--a domain between codons 1 and 58 is essential for the mitogen activation of S6K and confers rapamycin sensitivity; a second domain between codons 58 and 77 confers autoinhibition. We propose a model for the activation of S6 kinase in which mitogen-stimulated cellular factors interact with the amino terminus to negate the effects of the autoinhibitory domain.
Mol Cell Biol 1996 Jan
PMID:Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. 852 22


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