The <i>in Vitro</i> Phosphorylation of P53 by Calcium‐Dependent Protein Kinase C

Delphi, Christian; Huang, Kuo‐Ping; Scotto, Christian; Chapel, Agnès; Vincon, Mathilde; Chambaz, E.M.; Garin, Jérôme; Baudier, Jacques

doi:10.1111/j.1432-1033.1997.t01-1-00684.x

Cited by 35 publications

(33 citation statements)

References 34 publications

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“…The binding site of the casein kinase 2 (residues 325 to 344) (Gotz et al, 1999), the Ca 2+ -dependent protein kinase C (residues 320 to 346) (Delphin et al, 1997) and the adenovirus E4orf6 protein (residues 318 to 360) (Dobner et al, 1996) have been directly mapped within the TD. For other proteins, such as RelA (binding site between residues 300 and 393) (Ikeda et al, 2000) and hepatitis B virus HBx (binding site between residues 300 and 393) (Lin et al, 1997), the binding site was located within the p53 C-terminus.…”

Section: Oncogenementioning

confidence: 99%

The role of tetramerization in p53 function

2001

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Section: Oncogenementioning

confidence: 99%

The role of tetramerization in p53 function

2001

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“…Like its cognate peptide on MARCKS, O1 peptide is phosphorylated by PKC in vitro on multiple sites (30). Because binding of S100B to p53 inhibits p53 phosphorylation by PKC (16), we analyzed the effect of S100B on PKC-mediated phosphorylation of O1.…”

Section: S100b Protects P53 From Thermal Denaturation-heatingmentioning

confidence: 99%

“…Protein Kinase C Activity Assay and PKC Inhibitor Assay-The PKC assays were performed as described previously (30). To analyze the effect of S100B and other calcium-binding proteins on the inhibitor capacity of YF-O2 or W-O2 peptides, the following procedure was used.…”

Section: Peptides-the P53mentioning

confidence: 99%

“…10 l of PKC was subsequently added to each tube to reach a final concentration of 3 nM, and the protein mixture was vortexmixed. The phosphorylation reaction was immediately initiated by the addition of 20 l of a mixture containing O1 peptide used as substrate at 8 M final concentration plus MgCl 2 , phosphatidylserine, dioleoylglycerol, and [␥- 32 P]ATP at final concentrations used in standard PKC assay (30).…”

Section: Peptides-the P53mentioning

confidence: 99%

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Calcium-dependent Interaction of S100B with the C-terminal Domain of the Tumor Suppressor p53

Delphin

Ronjat

Deloulme

et al. 1999

Journal of Biological Chemistry

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In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53 1-320 ) and C-terminal (p53 ) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319 -393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 ؎ 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1357-381 (residues 357-381 in mouse p53) and YF-O2 320 -346 (residues 320 -346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.The S100 family is one of the largest subfamily of calciumbinding proteins that are thought to play roles in mediating calcium signals during cell growth, differentiation, and motility (reviewed in Ref. 1). These proteins are characterized by highly conserved helix-loop-helix calcium-binding domains, known as EF-hand motifs. The S100B is found in astroglial cells in the central nervous system but also in a number of tissues outside the nervous system, including adipose tissue, testis, skin, and lymphocytes (2). In cultured cells, synthesis of S100B is tightly regulated and is maximum in the G 1 phase of the cell cycle (3-5). The S100B protein is a noncovalent homodimer formed by the association of two S100-␤ subunits (6, 7). Calcium binding induces conformational changes in the protein structure (8 -10), destabilizing the S100B quaternary structure and allowing interaction with target proteins (7-12). For example, the Ca 2ϩ -dependent binding of S100B to microtubules (13), GFAP (14), Ndr protein kinase (15), and p53 (16) has been demonstrated. The identification of S100B target proteins is essential to better understand the mechanisms underlying S100B functions. Like calmodulin, S100B is likely to regulate multiple target proteins.The tumor suppressor p53 protein (17) is a putative intracellular target for the S100B protein. In vitro, S100B binds to p53 and inhibits p53 aggregation and phosphorylation by PKC (16).1 S100B also binds to a peptide derived from the extreme C-terminal end of p53 (12,18). A functional interaction between S100B and p53 was recently demonstrated in p53-negative mouse embryo fibroblast...

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“…The increase in p53-mediated transcriptional activity may be because of elevated levels of p53 in the cell (6,16) or increased sequence-specific binding ability (17,18). Post-translational modifications of p53, including phosphorylation by S and G 2 /M phase cyclin-dependent kinase-cyclin complexes (19,20), DNA-dependent protein kinase (21), protein kinase C (22), and casein kinase II (23), as well as C-terminal acetylation (24) have been found to enhance sequence-specific DNA binding in vitro. Furthermore, p53 has been shown to be acetylated at its C terminus after exposing cells to ultraviolet or ionizing radiation (18).…”

mentioning

confidence: 99%

High Affinity Insertion/Deletion Lesion Binding by p53

Szak

Pietenpol²

1999

Journal of Biological Chemistry

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In addition to binding DNA in a sequence-specific manner, p53 can interact with nucleic acids in a sequence-independent manner. p53 can bind short singlestranded DNA and double-stranded DNA containing nucleotide loops; these diverse associations may be critical for p53 signal transduction. In this study, we analyzed p53 binding to DNA fragments containing insertion/deletion mismatches (IDLs). p53 required an intact central domain and dimerization domain for high affinity complex formation with IDLs. In fact, the C terminus of p53 (amino acids 293-393) was functionally replaceable with a foreign dimerization domain in IDL binding assays. From saturation binding studies we determined that the K D of p53 binding to IDLs was 45 pM as compared with a K D of 31 pM for p53 binding to DNA fragments containing a consensus binding site. Consistent with these dissociation constants, p53-IDL complexes were dissociated with relatively low concentrations of competitor consensus site-containing DNA. Although p53 has a higher affinity for DNA with a consensus site as compared with IDLs, the relative number and availability of each form of DNA in a cell immediately after DNA damage may promote p53 interaction with DNA lesions. Understanding how the sequence-specific and nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how signaling cascades are initiated after DNA damage.

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The in Vitro Phosphorylation of P53 by Calcium‐Dependent Protein Kinase C

Cited by 35 publications

References 34 publications

The role of tetramerization in p53 function

The role of tetramerization in p53 function

Calcium-dependent Interaction of S100B with the C-terminal Domain of the Tumor Suppressor p53

High Affinity Insertion/Deletion Lesion Binding by p53

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