Eric I. Campos

Biological functions of the ING family tumor suppressors

Abstract.Early studies of the inhibitor of growth 1 (ING1) gene, the founding member of the ING tumor suppressor family, demonstrated that this gene plays an important role in apoptosis and cellular senescence. Four other related genes have since been identified and found to be involved in various biological activities, including cell cycle arrest, regulation of gene transcription, DNA repair and apoptosis. The biochemical functions of ING proteins as histone acetyltransferases and histone deacetylase co-factors ties this new tumor suppressor family to the regulation of transcription, cell cycle check-points, DNA repair and apoptosis. This review is aimed at summarizing the known biological functions of the ING tumor suppressors and the signalling pathways that they involve.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33ING1b

The tumour suppressor p33ING1b (ING1b for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell‐cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33ING1b phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half‐life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33ING1b with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33ING1b. We found that p33ING1b is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33ING1b. Furthermore, ultraviolet irradiation induces p33ING1b phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.

Phosphorylation of the tumor suppressor p33ING1b at Ser-126 influences its protein stability and proliferation of melanoma cells

ING (inhibitor of growth) tumor suppressors regulate cell‐cycle checkpoints, apoptosis, and ultimately tumor suppression. Among the ING family members, p33ING1b is the most intensively studied and plays an important role in the cellular stress response to DNA damage. Here we demonstrate that there is basal phosphorylation of p33ING1b at Ser‐126 in normal physiological conditions and that this phosphorylation is increased on DNA damage. The mutation of Ser‐126 to alanine dramatically shortened the half‐life of p33ING1b. Furthermore, we found that both Chk1 and Cdk1 can phosphorylate this residue. Interestingly, while Cdk1 can phosphorylate p33ING1b at Ser‐126 in nonstress conditions, Chk1 predominantly phosphory‐lates this residue on DNA damage, which suggests that p33ING1b is a downstream target of the ATM/ATR response cascade to genotoxic stress. More importantly, our data indicate that the Ser‐126 residue plays a key role in regulating the expression of cyclin B1 and proliferation of melanoma cells.—Garate, M., Campos, E. I., Bush, J. A., Xiao, H., Li, G. Phosphorylation of the tumor suppressor p33ING1b at Ser‐126 influences its protein stability and proliferation of melanoma cells. FASEB J. 21, 3705–3716 (2007)

Development of a PAN‐Specific, Affinity‐Purified Anti‐acetylated Lysine Antibody for Detection, Identification, Isolation, and Intracellular Localization of Acetylated Protein

Abstract Acetylation on the lysine residue is an important event of posttranslational modification of proteins. In this study, we developed a simple method to produce and to affinity purify the specific anti‐acetylated lysine polyclonal antibody, which is useful for the detection, identification, isolation, and intracellular localization of acetylated proteins on the lysine residues. We utilized the chemically acetylated hemocyanin of keyhole limpets (KLH) as an immunogen to raise the immune serum and to isolate the population of the acetylated lysine specific antibody using the immobilized acetylated lysine as immunoaffinity‐ligand. The isolated antibody was tested to be useful for ELISA, immunoblotting detection, immunofluorescent localization, and affinity isolation of the acetylated proteins.

Generation of a Polyclonal Antibody Specifically Against the p33ING1b Tumor Suppressor

Abstract The p33ING1b tumor suppressor protein plays a prominent role in cellular stress responses including cell cycle arrest, DNA repair, apoptosis, and chromatin remodeling. As the main product of the inhibitor of growth 1 (ING1) gene, p33ING1b is the most intensively studied protein of the ING family. So far, most ING1 antibodies have been raised against full‐length proteins. Since all ING1 isoforms share an identical carboxyl‐terminus, and commercially available ING1 antibodies often lack specificity, we sought to develop a polyclonal antibody capable of specifically recognizing the p33ING1b protein. Here, we describe the development and characterization of the p33ING1b‐specific antibody.