Kazuyasu Sakaguchi

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF‐7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co‐expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53‐mediated and UV‐induced apoptosis, and the reduction correlated with total abrogation of UV‐induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV‐induced apoptosis in a p53‐dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N‐terminal phosphorylation that regulates p53‐mediated apoptosis after UV radiation.

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage

Damage to DNA in the cell activates the tumour-suppressor protein p53 (ref. 1), and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53 (refs 2, 3), but the evidence for its involvement in this pathway is controversial,. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.

Human p53 Is Phosphorylated on Serines 6 and 9 in Response to DNA Damage-inducing Agents

To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser6 using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic,l-2-amino-4-phosphono-4,4-difluorobutanoic acid (F2Pab), in place of phosphoserine. Fmoc-F2Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F2Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1–39) peptide phosphorylated only at Ser6 but not the unphosphorylated peptide or the same peptide phosphorylated at Ser9, Ser15, Ser20, Ser33, or Ser37. Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser6 that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser9 using antibodies raised against a conventional phosphopeptide. Ser9 was phosphorylated by casein kinase 1in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F2Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T Helper Epitopes (PADRE) for antibody responses in vivo

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes.

ABSTRACT Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas

A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.

Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.

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

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 (p531–320) and C-terminal (p5373–393) 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-O2320–346 (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.

Interhelical angles in the solution structure of the oligomerization domain of p53: correction

tasks are described by L. Kaufman, S. Curtis, J. Z. Wang, and S. J. Williamson [Electroencephalogr. Clin. Neurophysiol. 82, 266 (1992)] and M. Fahle, J. Albrecht, H. Buelthoff, and D. Braun [Soc. Neurosci. Abstr. 20, 319 (1994)]. 22. K. Nakamura, A. Mikami, K. Kubota, Neuroreports 3, 117 (1992). 23. Pyramidal cells are modeled as identical integrateand-fire neurons. The membrane potential for each pyramidal cell is given by

Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element.

ABSTRACT ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-γ) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-γ-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-γ-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-γ-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-γ. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-γ-dependent gene regulation in an immune system-specific manner.