A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF) with approximately 5000 samples analyzed from. One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases - Steve Shears Identification of posttranslational modification of proteins that may be involved in DNA replication and repair - Sam Wilson and Bob London Identification of proteins in the BAF complexes under a variety of tissue types and/or conditions - Trevor Archer Analyses of histone variants - Serena Dudek Glucocorticoid receptor modifications and binding partners - John Cidlowski The core also is performing value added research in affinity techniques (Bromodoamin Enrichment)to aid in protein and PTM identifications. Other published projects or projects with manuscriptis in preparation include: Identification of RhoA binding partners: Arachidonic acid (AA) stimulates cell adhesion through a p38 MAPK-mediated RhoA signaling pathway. We performed a proteomic screen following AA-treatment identified nucleolin, a multifunctional nucleolar protein, in a complex with the GTPase, RhoA. AA-stimulated cell adhesion was blocked by expression of nucleolin-targeted shRNA. Furthermore, formation of a RhoA/ROCK/p38/nucleolin complex was blocked by expression of dominant negative RhoA. AA-treatment also induced ROCK-dependent serine phosphorylation of nucleolin and translocation of nucleolin from the nucleus to the cytoplasm, where it co-localized with RhoA. These data suggest a new signaling pathway through which the location and post-translational state of nucleolin are modulated. Phosphorylation of CD34: CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types and has been described as a marker for epidermal stem cells in mouse hair follicles. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C;however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309382) following PKC treatment. For this work, we used a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34. -Ken Tomer Macrophage Palmitoylation: S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine vs. mock treatment conditions. Of 1,183 proteins identified in 4 independent experiments, 80 proteins were significant for S-acylation at false discovery rate(FDR)=0.05, and 101 significant at FDR=0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and 3 presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. -Mike Fessler and Ken Tomer LDHC: Germ cell-specific lactate dehydrogenase C gene (Ldhc) leads to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. -Mitch Eddy Additional projects that have required more than negligible resources include efforts performed with the Armstrong, Birnbaumer, Blackshear, Eling, Freedman, Hall, Hu, and Negishi laboratories.
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