The Drosophila gene groucho (gro) encodes a transcriptional corepressor that has critical roles in many development processes. In an effort to illuminate the mechanism of Gro-mediated repression, we have employed Gro as an affinity reagent to purify Gro-binding proteins from embryonic nuclear extracts. One of these proteins was found to be the histone deacetylase Rpd3. Protein-protein interaction assays suggest that Gro and Rpd3 form a complex in vivo and that they interact directly via the glycine/proline rich (GP) domain in Gro. Cell culture assays demonstrate that Rpd3 potentiates repression by the GP domain. Furthermore, experiments employing a histone deacetylase inhibitor, as well as a catalytically inactive form of Rpd3, imply that histone deacetylase activity is required for efficient Gro-mediated repression. Finally, mutations in gro and rpd3 have synergistic effects on embryonic lethality and pattern formation. These findings support the view that Gro mediates repression, at least in part, by the direct recruitment of the histone deacetylase Rpd3 to the template, where it can modulate local chromatin structure. They also provide evidence for a specific role of Rpd3 in early development.

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.

Pim-1 kinase is a member of a distinct class of serine/ threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a nonconserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5-adenylyl-␤,␥-imidodiphosphate) complex to 2.1-Å resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.

No abstract

The IKK␤ and NEMO/IKK␥ subunits of the NF-B-activating signalsome complex are known to be essential for activating NF-B by inflammatory and other stresslike stimuli. However, the IKK␣ subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-B-dependent and -independent functions. In contrast to this generally accepted view of IKK␣'s physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKK␤ and NEMO/IKK␥, IKK␣ is also a global regulator of tumor necrosis factor ␣-and IL-1-responsive IKK signalsome-dependent target genes including many known NF-B targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, ␤ 2 -microglobulin, IL-1␣, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-B-dependent target genes were preferentially dependent on IKK␣ or IKK␤. Constitutive expression of a trans-dominant I B␣ superrepressor (I B␣SR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-B. A subset of NF-B target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-B in established MEFs. Overall, a sizable number of novel NF-B/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-␤ and -␦, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (␣ and ␤) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-B/IKK-induced gene, suggesting that IKKmediated NF-B activation can coordinately illicit negative effects on STAT signaling.The NF-B transcription factors are pivotal regulators of gene expression programs culminating in stress-like responses and the genesis of innate and acquired immunity (reviewed in Refs. 1-4). A host of extracellular stimuli including inflammatory cytokines, viral and bacterial infections, oxidative and DNA-damaging agents, UV light, and osmotic shock can all result in NF-B activation (1, 3-5). NF-B transcription factors bind to DNA as hetero-or homodimers that are selectively derived from five possible subunits (RelA/p65, c-Rel, RelB, p50, and p52) with each binding to half of a conserved 10-base pair consensus sequence (GGGRNWTYCC) (1, 5). Whereas the RelA/p65 and p50 subunits are ubiquitously expressed, the p52, c-Rel, and RelB subunits are more functionally important in specific differentiated cell types (1, 6). Cytoplasmic p50/p65 heterodimers, c-Rel homodimers, and RelB are bound to I Bs (inhibitors of NF-B...