Recruitment of ikaros to pericentromeric heterochromatin is regulated by phosphorylation.

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.

Global analysis of protein palmitoylation in yeast.

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore midgut.

The plant hormone jasmonic acid (JA) activates host defense responses against a broad spectrum of herbivores. Although it is well established that JA controls the expression of a large set of target genes in response to tissue damage, very few gene products have been shown to play a direct role in reducing herbivore performance. To test the hypothesis that JA-inducible proteins (JIPs) thwart attack by disrupting digestive processes in the insect gut, we used a MS-based approach to identify host proteins that accumulate in the midgut of Manduca sexta larvae reared on tomato (Solanum lycopersicum) plants. We show that two JIPs, arginase and threonine deaminase (TD), act in the M. sexta midgut to catabolize the essential amino acids Arg and Thr, respectively. Transgenic plants that overexpress arginase were more resistant to M. sexta larvae, and this effect was correlated with reduced levels of midgut Arg. We present evidence indicating that the ability of TD to degrade Thr in the midgut is enhanced by herbivore-induced proteolytic removal of the enzyme's C-terminal regulatory domain, which confers negative feedback regulation by isoleucine in planta. Our results demonstrate that the JA signaling pathway strongly influences the midgut protein content of phytophagous insects and support the hypothesis that catabolism of amino acids in the insect digestive tract by host enzymes plays a role in plant protection against herbivores.

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease.

Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.

The crystal structure of Pichia pastoris lysyl oxidase.

Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.

Crystallization of Pichia pastoris lysyl oxidase.

A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms. PPLO is a glycoprotein. The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa). Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution. Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution. Native data have been recorded from each crystal form at 100 K using synchrotron radiation. A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit.

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.