Protein Microcharacterization

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A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF). 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 Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases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 protiens that may be involved in DNA replication and repair Sam Wilson Identification and semi-quantitative analyses of proteins in the CA1, CA3, and DG regions of the brains of rats treated with trimethyl-tin (TMT) G. Jean Harry Identification of proteins in the BAF complexes under a variety of tissue types and/or conditions Trevor Archer Identification of proteins that have altered affinity to mutants of the glucocorticoid receptor John Cidlowski The core also is performing value added research in separations (off-gel electrophoresis) and 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 Adam 9 substrates: Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxy fluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)- NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)- NH2, C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)- NH2, and CD23, Dabcyl-Leu-Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2. Cleavage sites were effectively processed by ADAM9 and turnover numbers determined. The valine containing precursor TNF-alpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed the most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high-throughput screening assays for ADAM9. Bob Petrovich Phosphorylation of SIRT1: SIRT1, an NAD+-dependent protein deacetylase, plays an important role in a variety of physiological processes including stress response and energy metabolism. We have demonstrated that SIRT1 and anti-apoptotic members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family, DYRK1A and DYRK3, directly phosphorylate SIRT1 at the Threonine 522 residue. DYRK-mediated phosphorylation of Thr522 increases SIRT1 activity, resulting in deacetylation and inhibition of p53 upon DNA damage. Xiaoling Li Phosphorylation of TTP in the presence of CIN85: Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that is considered to be an anti-inflammatory protein. We found that co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. These data demonstrate that CIN85 binding to human TTP leads to increased phosphorylation of hTTP, possibly through association with MEKK4, with potential but unknown functional consequences. Perry Blackshear Identification and Characterization of AChBP: We identified a homolog of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella capitata, from the annelid phylum. Mass spectrometry results indicate that Asn122 and Asn216 of cc-AChBP are glycosylated when expressed using HEK293 cells. Small angle X-ray scattering (SAXS) data suggest that the overall shape of cc-AChBP is similar to homologs with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and bungarotoxin bind to cc-AChBP with high affinity, and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. Jerry Yakel Additional projects that have required more than negligible resources include efforts performed with the Armstrong, Birnbaumer, Eling, Hall, Hu, and Resnick, laboratories.