Biomedical subjects

J DiDonato

Publications and source records attributed to J DiDonato.

3 recordsLinked to original sources

PubMed1999

An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis.

To examine the role of nuclear factor (NF)-kappaB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor kappaB-alpha (IkappaB-alphaA32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant [m]IkappaB-alpha mice). Thymocyte development proceeded normally in the mIkappaB-alpha mice. However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals. The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4(+)CD8(+); DP) thymocytes from the mIkappaB-alpha mice were resistant to alpha-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by gamma-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIkappaB-alpha thymocytes maintained high level expression of bcl-xL after alpha-CD3 treatment. Taken together, these results demonstrated important roles for NF-kappaB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-kappaB is required for the alpha-CD3-mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.

Animals↗

PubMed1996

Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.

Extracellular stimuli that activate the transcription factor NF-kappaB cause rapid phosphorylation of the IkappaBalpha inhibitor, which retains NF-kappaB in the cytoplasm of nonstimulated cells. Phosphorylation of IkappaBalpha is followed by its rapid degradation, the inhibition of which prevents NF-kappaB activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IkappaBalpha. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IkappaBalpha degradation. Substitutions of the homologous sites in IkappaBbeta, serines 19 and 23, also prevent inducible IkappaBbeta degradation. We suggest that activation of a single IkappaB kinas e or closely related IkappaB kinases is the first cr itical step in NF-kappaB activation. Once phosphorylated, IkappaB is ubiquitinated. Unlike wild-type IkappaBalpha, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IkappaBalpha without affecting its phosphorylation, polyubiquitination is required for inducible IkappaB degradation.

3T3 Cells↗

PubMed1992

Herpes simplex virus type 1 polypeptide ICP4 bends DNA.

ICP4, the major regulatory polypeptide of herpes simplex virus type 1, is expressed at the earliest stages of virus infection and is required for the activation of transcription from the majority of viral promoters. It is a DNA binding protein which specifically recognises bipartite sites related to the sequence ATCGTnnnnnCGG. In this report we show that both partially purified ICP4, and its isolated DNA binding domain, bend DNA at occupied binding sites. The apparent angles of bend at two different binding sites were very similar and in both cases the centre of the bend was very close to the binding site sequence.

Binding Sites↗