ABSTRACT
The versatile phosphoprotein pp32 is involved in important physiological processes, including cell proliferation, apoptosis, mRNA transport, and transcription. We have previously reported that pp32, through histone masking, inhibits histone acetylation and transcriptional activation by histone acetyltransferases. However, how pp32 itself is regulated remained largely unknown. Although pp32 is a phosphoprotein, neither the phosphorylation sites nor the cellular kinase has been identified. In this report, utilizing an in vitro kinase assay and a biochemical purification scheme, we identify casein kinase II as a cellular pp32-kinase. Our deletion and site-specific mutagenesis studies identify serines 158 and 204 as the sites of phosphorylation. Generation and utilization of antibodies with higher affinity for phospho-pp32 demonstrate that pp32 is indeed phosphorylated in vivo at these two sites. Mutagenesis studies on pp32 suggest a role for serines 158 and 204 in its function. The identification of the pp32 kinase and the sites of pp32 phosphorylation as well as the generation of antibodies with higher affinity for phospho-pp32 should now provide key information and tools for future studies on pp32 regulation.
Subject(s)
Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/isolation & purification , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Casein Kinase II , Cell Nucleus/enzymology , Dichlororibofuranosylbenzimidazole/chemistry , Enzyme Inhibitors/chemistry , HeLa Cells , Heparin/chemistry , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphoproteins/genetics , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Sequence Deletion , Serine/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , TransfectionABSTRACT
Various post-translational modifications of histones significantly influence gene transcription. Although un- or hypoacetylated histones are tightly linked to transcriptional repression, the mechanisms and identities of chromatin signal transducer proteins integrating histone hypoacetylation into repression in humans have remained largely unknown. Here we show that the mammalian histone-binding proteins and inhibitor of acetyltransferases (INHAT) complex subunits, Set/template-activating factor-Ibeta (TAF-Ibeta) and pp32, specifically bind to unacetylated, hypoacetylated, and repressively marked histones but not to hyperacetylated histones. Additionally, Set/TAF-Ibeta and pp32 associate with histone deacetylases in vitro and in vivo and repress transcription from a chromatin-integrated template in vivo. Finally, Set/TAF-Ibeta and pp32 associate with an endogenous estrogen receptor-regulated gene, EB1, in the hypoacetylated transcriptionally inactive state but not with the hyperacetylated transcriptionally active form. Together, these data define a novel in vivo mechanistic role for the mammalian Set/TAF-Ibeta and pp32 proteins as transducers of chromatin signaling by integrating chromatin hypoacetylation and transcriptional repression.
Subject(s)
Chromatin/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Histones/chemistry , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Transcription Factors/chemistry , Acetylation , Cell Line , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/physiology , DNA-Binding Proteins , Estrogens/metabolism , Glutathione Transferase/metabolism , HeLa Cells , Histone Chaperones , Histones/metabolism , Humans , Ligands , Luciferases/metabolism , Models, Biological , Nuclear Proteins/physiology , Peptides/chemistry , Phosphoproteins/physiology , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Signal Transduction , Transcription Factors/physiology , Transcription, GeneticABSTRACT
The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.
