Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53

1992 ◽  
Vol 12 (11) ◽  
pp. 5041-5049
Author(s):  
S P Lees-Miller ◽  
K Sakaguchi ◽  
S J Ullrich ◽  
E Appella ◽  
C W Anderson
Keyword(s):  
Protein Kinase ◽  
Synthetic Peptides ◽  
Wild Type Mouse ◽  
Tumor Suppressor Protein ◽  
Transactivation Domain ◽  
Wild Type ◽  
Amino Terminal ◽  
Human Dna ◽  
Serine 392 ◽  
P53 Peptides

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.

scholarly journals Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

1992 ◽  
Vol 12 (11) ◽  
pp. 5041-5049 ◽  
Author(s):  
S P Lees-Miller ◽  
K Sakaguchi ◽  
S J Ullrich ◽  
E Appella ◽  
C W Anderson
Keyword(s):  
Protein Kinase ◽  
Synthetic Peptides ◽  
Wild Type Mouse ◽  
Tumor Suppressor Protein ◽  
Transactivation Domain ◽  
Wild Type ◽  
Amino Terminal ◽  
Human Dna ◽  
Serine 392 ◽  
P53 Peptides

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.

Abstract 1493: Erythropoietin Stimulates Endothelial Biosynthesis of Tetrahydrobiopterin by Activation of Phosphatidylinositol 3-kinase/Protein Kinase B Signal Transduction Pathway

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Livius V d’Uscio ◽  
Anantha V Santhanam ◽  
Zvonimir S Katusic
Keyword(s):  
Protein Kinase ◽  
Enzymatic Activity ◽  
Vascular Endothelium ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Protein Kinase B ◽  
Wild Type Mouse ◽  
Protective Effects ◽  
Wild Type ◽  
Deficient Mice

Erythropoietin (EPO) has been recognized as a tissue protective cytokine. Recently, it has been shown that vascular protective effects of EPO are dependent on activation of endothelial nitric oxide synthase (eNOS). Tetrahydrobiopterin (BH 4 ) is an essential cofactor required for enzymatic activity of eNOS. Therefore, our objective was to characterize the effect of EPO on biosynthesis of BH 4 in vascular wall. Incubation of isolated wild-type (C57BL/6J) mouse aortas for 18 hours at 37°C in minimal essential medium supplemented with recombinant human EPO (1–50 U/ml) caused concentration-dependent increase in intracellular levels of BH 4 as determined by HPLC analysis. Maximum biosynthesis of BH 4 was detected at therapeutic concentrations of 5 U/mL (15.8±1.3 pmol/mg protein; P<0.05 vs control: 8.2±0.4 pmol/mg protein; n=6 – 8). Oxidative products of BH 4 , 7,8-dihydrobiopterin, were unaffected by EPO indicating that EPO does not affect oxidation of BH 4 . Removal of the endothelium abolished EPO-induced biosynthesis of BH 4 (P<0.05; n=5) demonstrating that the vascular endothelium is a major source of BH 4 . Treatment of intact isolated wild-type mouse aortas with a selective phosphatidylinositol (PI)3-kinase inhibitor wortmannin (1 μM) significantly reduced BH 4 biosynthesis by EPO (8.4±0.6 pmol/mg protein; P<0.05; n=6). Stimulatory effect of EPO on production of BH 4 in aorta was also detected in wild-type mice treated with recombinant human EPO (1000 U/kg, s.c. biweekly) for 14 days (P<0.05; n=6). This vascular effect was abolished in protein kinase B (Akt) 1-deficient mice treated with EPO (P<0.05; n=5). Furthermore, aortic GTP cyclohydrolase I (GTPCH I) enzymatic activity was augmented in EPO treated wild-type mice (0.48±0.12 pmol neopterin/mg protein; P<0.05 vs control: 0.23±0.05 pmol neopterin/mg; n=4) but not in EPO treated Akt 1-deficient mice (0.21±0.03 pmol neopterin/mg; n=4), indicating that the selective increase in BH 4 levels was caused by de-novo biosynthesis of BH 4 via Akt/GTPCH I pathway. Our results demonstrate that EPO stimulates biosynthesis of BH 4 in vascular endothelium. This effect is most likely designed to provide optimal intracellular concentration of cofactor necessary for EPO-induced elevation of eNOS activity.

scholarly journals γ-Catenin is regulated by the APC tumor suppressor and its oncogenic activity is distinct from that of β-catenin

2000 ◽  
Vol 14 (11) ◽  
pp. 1319-1331 ◽  
Author(s):  
Frank T. Kolligs ◽  
Barbara Kolligs ◽  
Karen M. Hajra ◽  
Gang Hu ◽  
Masachika Tani ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Wnt Signaling ◽  
Target Genes ◽  
Wnt Signaling Pathway ◽  
Tumor Suppressor Protein ◽  
Wild Type ◽  
Downstream Target ◽  
Amino Terminal ◽  
Transforming Activity ◽  
Altered Regulation

β-Catenin and γ-catenin (plakoglobin), vertebrate homologs of Drosophila armadillo, function in cell adhesion and the Wnt signaling pathway. In colon and other cancers, mutations in the APC tumor suppressor protein orβ-catenin's amino terminus stabilizeβ-catenin, enhancing its ability to activate transcription of Tcf/Lef target genes. Thoughβ- and γ-catenin have analogous structures and functions and like binding to APC, evidence that γ-catenin has an important role in cancer has been lacking. We report here that APC regulates bothβ- and γ-catenin andγ-catenin functions as an oncogene. In contrast to β-catenin, for which only amino-terminal mutated forms transform RK3E epithelial cells, wild-type and several amino-terminal mutated forms of γ-catenin had similar transforming activity. γ-Catenin's transforming activity, like β-catenin's, was dependent on Tcf/Lef function. However, in contrast toβ-catenin, γ-catenin strongly activated c-Myc expression and c-Myc function was crucial for γ-catenin transformation. Our findings suggest APC mutations alter regulation of bothβ- and γ-catenin, perhaps explaining why the frequency of APC mutations in colon cancer far exceeds that of β-catenin mutations. Elevated c-Myc expression in cancers with APC defects may be due to altered regulation of both β- andγ-catenin. Furthermore, the data implyβ- and γ-catenin may have distinct roles in Wnt signaling and cancer via differential effects on downstream target genes.

scholarly journals Functional Anatomy of Herpes Simplex Virus 1 Overlapping Genes Encoding Infected-Cell Protein 22 and US1.5 Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4305-4315 ◽  
Author(s):  
William O. Ogle ◽  
Bernard Roizman
Keyword(s):  
Protein Kinase ◽  
Infected Cell ◽  
Functional Anatomy ◽  
Cell Protein ◽  
Amino Terminus ◽  
Posttranslational Processing ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Amino Terminal ◽  
Infected Cell Protein

ABSTRACT Earlier studies have shown that (i) the coding domain of the α22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore ΔUS1.5 are avirulent and fail to express normal levels of subsets of both α (e.g., ICP0) or γ2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3′-terminal codons of ICP22 and US1.5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1.5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3′-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.

scholarly journals Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic Adenosine Monophosphate–Dependent Protein Kinase

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3193-3201 ◽  
Author(s):  
Darren Casteel ◽  
Modem Suhasini ◽  
Tanima Gudi ◽  
Reza Naima ◽  
Renate B. Pilz
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Transactivation Domain ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Murine Erythroleukemia

Abstract Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase–deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser169; Ala substitution for Ser169 resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore β-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser169 → Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.

scholarly journals Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic Adenosine Monophosphate–Dependent Protein Kinase

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3193-3201 ◽  
Author(s):  
Darren Casteel ◽  
Modem Suhasini ◽  
Tanima Gudi ◽  
Reza Naima ◽  
Renate B. Pilz
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Transactivation Domain ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Murine Erythroleukemia

Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase–deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser169; Ala substitution for Ser169 resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore β-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser169 → Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.

scholarly journals Functional Interaction of the Bovine Papillomavirus E2 Transactivation Domain with TFIIB

1998 ◽  
Vol 72 (2) ◽  
pp. 1013-1019 ◽  
Author(s):  
Jun-Mei Yao ◽  
David E. Breiding ◽  
Elliot J. Androphy
Keyword(s):  
Dna Binding ◽  
Transcriptional Activity ◽  
Binding Domain ◽  
Limiting Factors ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Wild Type ◽  
Functional Interaction ◽  
E2 Protein ◽  
Amino Terminal

ABSTRACT Induction of gene expression by the papillomavirus E2 protein requires its ∼220-amino-acid amino-terminal transactivation domain (TAD) to interact with cellular factors that lead to formation of an activated RNA polymerase complex. These interaction partners have yet to be identified and characterized. The E2 protein localizes the transcription complex to the target promoter through its carboxy-terminal sequence-specific DNA binding domain. This domain has been reported to bind the basal transcription factors TATA-binding protein and TFIIB. We present evidence establishing a direct interaction between amino acids 74 to 134 of the E2 TAD and TFIIB. Within this region, the E2 point mutant N127Y was partially defective and W99C was completely defective for TFIIB binding in vitro, and these mutants displayed reduced or no transcriptional activity, respectively, upon transfection into C33A cells. Overexpression of TFIIB specifically restored transactivation by N127Y to close to wild-type levels, while W99C remained inactive. To further demonstrate the functional interaction of TFIIB with the wild-type E2 TAD, this region was fused to a bacterial DNA binding domain (LexA:E2:1-216). Upon transfection with increasing amounts of LexA:E2:1-216, there was reduction of its transcriptional activity, a phenomenon thought to result from titration of limiting factors, or squelching. Squelching of LexA:E2:1-216, or the wild-type E2 activator, was partially relieved by overexpression of TFIIB. We conclude that a specific region of the E2 TAD functionally interacts with TFIIB.

Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger

1993 ◽  
Vol 264 (4) ◽  
pp. C944-C950 ◽  
Author(s):  
K. Takaichi ◽  
D. F. Balkovetz ◽  
E. Van Meir ◽  
D. G. Warnock
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Wild Type Mouse ◽  
Host Cells ◽  
Wild Type ◽  
Cytosolic Ph ◽  
Kinase C ◽  
In The Wild ◽  
Mouse Cells ◽  
The Hill

These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na(+)-H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na(+)-H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild-type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (Vmax) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the Vmax and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.

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