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S R Grossman

Publications and source records attributed to S R Grossman.

At least 19 recordsLinked to original sources
PubMed2006

hHR23B is required for genotoxic-specific activation of p53 and apoptosis.

Rad23 proteins function in both DNA repair and protein stability regulation. As ubiquitinated forms of p53 are stabilized after DNA damage in concert with p53 functional activation, and human Rad23 proteins (hHR23A and B) regulate p53 stability in unstressed cells, the role of hHR23B in post-genotoxin regulation of p53 was investigated. Depletion of hHR23B by specific short interfering RNA before genotoxic exposure attenuated p53, p21 and bax induction, abrogated the accumulation of ubiquitinated p53 and suppressed apoptosis. Expression of ubiquitin derivatives with all lysines mutated except K48 or K63 demonstrated that K48-linked p53-ubiquitin conjugates were specifically induced after DNA damage. hHR23B, along with native and ubiquitinated p53, accumulated in chromatin after genotoxic exposure, and the accumulation of ubiquitinated p53 in chromatin was prevented by hHR23B depletion. Chromatin immunoprecipitation analysis demonstrated that hHR23B and p53 both localized to the p21 promoter shortly after DNA damage. hHR23B thus plays a critical role in the activation and function of p53 after specific genotoxic exposures.

Apoptosis↗
PubMed2001

Phosphorylation of the PTEN tail acts as an inhibitory switch by preventing its recruitment into a protein complex.

PTEN is a tumor suppressor protein that functions, in large part, by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate and by doing so antagonizing the action of phosphoinositide 3-kinase. PTEN structural domains include an N-terminal phosphatase domain, a lipid-binding C2 domain, and a 50-amino acid C-terminal tail that contains a PDZ binding sequence. We showed previously that phosphorylation of the PTEN tail negatively regulates PTEN activity. We now show that phosphorylated PTEN exists in a monomeric "closed" conformation and has low affinity for PDZ domain-containing proteins. Conversely, when unphosphorylated, PTEN is in an "open" conformation, is recruited into a high molecular weight complex (PTEN-associated complex), and strongly interacts with PDZ-containing proteins such as MAGI-2. As a consequence, when compared with wild-type PTEN, the phosphorylation-deficient mutant form of PTEN strongly cooperates with MAGI-2 to block Akt activation. These results indicate that phosphorylation of the PTEN tail causes a conformational change that results in the masking of the PDZ binding domain. Consequently, the ability of PTEN to bind to PDZ domain-containing proteins is reduced dramatically. These data suggest that phosphorylation of the PTEN tail suppresses the activity of PTEN by controlling the recruitment of PTEN into the PTEN-associated complex.

Activin Receptors, Type II↗
PubMed2001

p19ARF targets certain E2F species for degradation.

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Blotting, Northern↗
PubMed2001

p300/CBP/p53 interaction and regulation of the p53 response.

Substantial evidence points to a critical role for the p300/CREB binding protein (CBP) coactivators in p53 responses to DNA damage. p300/CBP and the associated protein P/CAF bind to and acetylate p53 during the DNA damage response, and are needed for full p53 transactivation as well as downstream p53 effects of growth arrest and/or apoptosis. Beyond this simplistic model, p300/CBP appear to be complex integrators of signals that regulate p53, and biochemically, the multipartite p53/p300/CBP interaction is equally complex. Through physical interaction with p53, p300/CBP can both positively and negatively regulate p53 transactivation, as well as p53 protein turnover depending on cellular context and environmental stimuli, such as DNA damage.

Acetylation↗
PubMed2000

Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter.

The Epstein-Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs). Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF. P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography. EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300. In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression. Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect. In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity. Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.

Acetyltransferases↗
PubMed2000

Cells degrade a novel inhibitor of differentiation with E1A-like properties upon exiting the cell cycle.

Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.

Acetyltransferases↗
PubMed1998

p300/MDM2 complexes participate in MDM2-mediated p53 degradation.

Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.

Cell Line↗
PubMed1997

Binding and modulation of p53 by p300/CBP coactivators.

The adenovirus E1A and SV40 large-T-antigen oncoproteins bind to members of the p300/CBP transcriptional coactivator family. Binding of p300/CBP is implicated in the transforming mechanisms of E1A and T-antigen oncoproteins. A common region of the T antigen is critical for binding both p300/CBP and the tumour suppressor p53, suggesting a link between the functions of p53 and p300. Here we report that p300/CBP binds to p53 in the absence of viral oncoproteins, and that p300 and p53 colocalize within the nucleus and coexist in a stable DNA-binding complex. Consistent with its ability to bind to p300, E1A disrupted functions mediated by p53. It reduced p53-mediated activation of the p21 and bax promoters, and suppressed p53-induced cell-cycle arrest and apoptosis. We conclude that members of the p300/CBP family are transcriptional adaptors for p53, modulating its checkpoint function in the G1 phase of the cell cycle and its induction of apoptosis. Disruption of p300/p53-dependent growth control may be part of the mechanism by which E1A induces cell transformation. These results help to explain how p53 mediates growth and checkpoint control, and how members of the p300/CBP family affect progression from G1 to the S phase of the cell cycle.

3T3 Cells↗
PubMed1996

EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein-Barr virus-transformed B lymphocytes.

Although genetic and biochemical data indicate that the cell protein RBPJkappa is a mediator of EBNA-2 and EBNA-3C effects on transcriptional regulatory elements, the extent of association of these Epstein-Barr virus nuclear proteins with RBPJkappa in transformed B lymphocytes has not been determined. We now report that most of the EBNA-2 and at least 20% of the EBNA-3C coimmunoprecipitated with RBPJkappa from extracts of transformed B lymphocytes that contained most of the cellular EBNA-2 and EBNA3C. Both proteins are associated preferentially with the smaller of the two RBPJkappa isoforms. EBNA-2-RBPJkappa complexes do not contain EBNA-3C, and EBNA-3C-RBPJkappa complexes do not contain EBNA-2. Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody. Promoters with RBPJkappa sites in their regulatory elements are likely to be differentially regulated by these RBPJkappa-EBNA-2 and RBPJkappa-EBNA-3 complexes.

Antigens, Viral↗
PubMed1995

Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1.

Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.

Antigens, Viral↗
PubMed1994

The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).

Amino Acid Sequence↗
PubMed1993

The tryptophan bridge is a critical feature of the papillomavirus E2 DNA binding domain.

The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.

Base Sequence↗
PubMed1993

Human papillomavirus type 18 E7 protein requires intact Cys-X-X-Cys motifs for zinc binding, dimerization, and transformation but not for Rb binding.

Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.

Adenovirus E1A Proteins↗
PubMed1992

Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target.

The dominant transcriptional regulator of the papillomaviruses, E2, binds to its specific DNA target through a previously unobserved dimeric antiparallel beta-barrel. The DNA is severely but smoothly bent over the barrel by the interaction of successive major grooves with a pair of symmetrically disposed alpha-helices. The specific interface is an 'interwoven' network of interactions where the identifying base pairs of the target contact more than one amino-acid side chain and the discriminating amino acids interact with more than one base pair.

Amino Acid Sequence↗
PubMed1992

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

Amino Acid Sequence↗
PubMed1991

Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences.

The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.

Baculoviridae↗
PubMed1990

Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein.

The introduction of the bovine (BPV) or human papillomavirus E6 gene into susceptible cells can result in their transformation, but there are few clues to the mechanism of action of the E6 gene. The characteristic features of E6 proteins are their small size (approximately 150 amino acids) and the potential to form two large zinc fingers. To determine if E6 can function as a transcription factor, the BPV E6 gene was fused to the sequence specific DNA binding peptide encoded by the BPV E2 gene. This chimeric E6-E2 protein trans-activated promoters that incorporated E2 binding elements in both rodent cells and Saccharomyces cerevisiae. In the absence of E6-E2 localization to the target promoter, trans-activation did not occur. Alteration of the cysteine residues at the base of each finger abrogated the transcriptional activity of the E6-E2 hybrids. These data demonstrated that the BPV E6 gene encodes a transcription activation domain and imply that a specific structure of the protein, most likely the zinc fingers, is critical for this function. Since these cysteine mutants are also transformation defective, E6 transcriptional functions may be required for its oncogenic activity.

Amino Acid Sequence↗