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Core Facilities

BIACORE

Clinical Immunology
Laboratory

Myositis Testing

DNA Sequencing

Flow Cytometry

Imaging Core Facility

Microarray Research
Facility

Signal Transduction
Core Facility

Small Animal MRI

Shared Facilities

Laboratory Animal
Resource Center (LARC)

Institutional Animal
Care and Use
Committee (IACUC)

 

  

The core facility will provide state of the art expertise and instrumentation to research projects at OMRF. A number of specific techniques are described below.

Services:

Measurements of intracellular calcium

Measurements of protein intreactions

Phosphoamino acid analysis

Kinase measurements

GTPase activation measurements

Transient transfections

References

Description of Services:

  1. Measurements of intracellular calcium:

    Changes in cytoplasmic calcium are a common feature of many signaling pathways, including those initiated by protein tyrosine kinases and by heterotrimeric GTP-binding proteins. The core lab is prepared for such measurements using Indo-1 fluorescence assessed by a Perkin-Elmer LS-50 spectrofluorimeter. Cells are loaded with Indo-1-acetoxymethyl ester, washed and suspended buffered saline. 1x106 cells are stimulated and/or treated with agents provided by the investigaotrs and changes in cytoplasmic calcium followed by Indo-1 fluorescence will be monitored using a spectrofluorimeter. Violet (unbound) and blue (Ca2+-bound) emission wavelengths will be assessed and the ratio will be used to measure cellular calcium levels, as our laboratory has described (1).

  2. Measurements of protein interactions:

    Protein associations mediated by numerous interaction domains are a fundamental and determining feature in signal transduction induced by receptors of hematopoietic cells. Accordingly, a number of techniques have evolved to assess the precise nature and identity of interacting proteins in various signaling situations. The signal transduction core facility has extensive experience in these techniques, some of which are listed below with documentation of their use. Invariably, however, immunoblotting of interacting proteins is the method of choice for their detection. The signal transduction core facility housed shared PAGE and electrophoretic transfer equipment, all materials for immunoblotting, and a Roche Lumilmager for immuno-based detection and quantitation of proteins and nucleic acids on filters.

    1. Protein domain pull-downs: Recombinant modular domains of proteins (SH2, SH3, PH, PTB, etc.) are made as fusion proteins of glutathione-S-transferase (GST), maltose-binding protein (MBP) or 6x-histidine (6xHis), expressed in either E. coli (2) or S. pombe (3). Personnel in the core facility have experience using both models of expression and of the various epitope tags that facilitate affinity purification. Large scale preparations (1-5 liters) are induced with IPTG (E. coli) or methanol (S. pombe) and purified in batch by incubation of lysates with the affinity matrix and bound proteins are washed and eluted. Recombinant proteins can be subjected to additional purification steps (size exclusion, ion exchange) if necessary, using a BioRad medium-pressure chromatography system, housed in the Core facility.

      The facility maintains a large reserve of various interaction domain molecules as GST-fusion proteins; each of these has been optimized for expression of soluble protein in E coli or S. pombe and used in functional studies. All of these existing constructs, as well as techniques for the generation of new constructs as needed, are available to OMRF investigators.

    2. Peptide and phosphopeptide pull-downs: Isolated SH2 (4, 5) and SH3 (6, 7) domains recognize defined linear amino acid sequences within their binding partners. We (8, 9) and others have exploited this feature of interaction modules within signaling proteins by using synthetic, biotinylated peptides in in vitro binding studies. Peptides are incubated in lysates derived from resting or activated cells and incubated overnight. The biotinylated peptide and its bound proteins are precipitated with streptavidin-Sepharose and analyzed by SDS/PAGE and immunoblot. The peptide core facility has experience in generating biotinylated phosphopeptides and will be instrumental in providing investigators with these reagents.

    3. Co-immunoprecipitations: Protein interactions identified by peptide and protein pull-down experiments will also been analyzed by co-immunoprecipitation. Cells will be stimulated and immunoprecipitated with antisera directed against defined targets and immunoblotted with antibodies against candidate interacting proteins. Individuals within the core facility have extensive and documented experience using this strategy to identify protein-protein interactions (9-11), and this experience will be shared with users of the facility.

    4. Far-western analysis: Evidence for direct protein-protein interactions (as opposed to indirect interactions involving an adapter protein) can be obtained by Far western blots. In these assays, a known protein is obtained by immunoprecipitation, separated from impurities by SDS/PAGE and transferred to filters. The filter is then probed with a labeled, recombinant interaction domain derived from the interacting partner in question. Binding can be detected by immunoblot of an epitope tag present on the probe, or by using a biotinylated probe followed by immunoblot with labeled streptavidin. Personnel within the core facility have used this technique, applying biotinylated phosphopeptides to SH2 domain-containing proteins that have been immobilized on filters (8).
       

  3. Phosphoamino acid analysis:

    Protein phosphorylated on Ser/Thr or Tyr residues can be detected by phosphoamino acid analysis, and the precise sites of phosphorylation can be determined by phosphopeptide analysis (12). The signal transduction core facility has purchased a Hunter Thin Layer Electrophoresis (TLE) apparatus for these types of assays. Experimentally, cells will be metabolically labeled with [32P]-inorganic phosphate and the protein of interest will be separated by SDS/PAGE followed by autoradiography. The appropriate band(s) will excised from the gel and either digested with HCl (for phosphoamino acid analysis) or with protease to generate labeled peptide fragments (for phosphopeptide analysis). The resulting digest will be separated using TLE and labeled amino acids or peptides will be detected by autoradiography. Labeled phosphoamino acids will be identified by co-migration with authentic commercial standards; phosphopeptides will require additional purification by HPLC and analysis by mass spectroscopy, or can be directly sequenced.
     

  4. Kinase measurements:

    Activation of tyrosine protein kinases is the most proximal event in signal transduction by immunoreceptors or growth factor receptors, and frequently leads to stimulation of other essential serine/threonine kinases. Thus, the induced catalytic activity of these kinases is often used as an indicator of receptor-triggered signal transduction. Generally, the assay is limited by identifying appropriate immunoprecipitation conditions, a useful in vitro substrate, and/or buffer and co-factor conditions that support the in vitro reaction. Fortunately, many of these limiting features have been resolved for numerous Ser/Thr and Tyr kinases, and the personnel in the core facility have considerable experience in determining these variables when novel kinases are identified.

    1. Ser/Thr kinases - MAP kinases, Protein kinase C, Akt: Personnel within the signal transduction core facility have described and published in vitro assays of Raf, MEK and MAP kinases (10, 13), protein kinase C (14,15), and the survival kinase, Akt (16). Briefly, the activity of these kinases is assessed by immunoprecipitation and incubation, with or without an artificial substrate, in the presence of [g32P]ATP. The autophosphorylated kinase or phosphorylated in vitro substrate is detected by autoradiography after SDS/PAGE and the kinase activity is determined by quantitating the phosphorylated material. The specificity of the assay is the result of the specificity of the immunoprecipitating reagent, and of the ability of the kinase to recognize and phosphorylate distinct in vitro substrates, or phosphorylate itself in autophosphorylation.

    2. Tyr kinase - family members of Src; Syk; JAK: Personnel within the signal transduction core facility have described and published assays of Src- and Syk-family protein tyrosine kinases (17-19); autophosphorylation of JAK kinases will be used as an indicator of kinase activation in cytokine-stimulated cells. These assays are performed and quantitated essentially as described above.

    3. Lipid kinase - PtdIns 3-kinase: Anti-phosphotyrosine (19) or anti-p85 immunoprecipitates (16) have been used by individuals in the signal transduction core facility as indicators of PtdIns 3-kinase activation. The precipitates are mixed with commercial PtdIns and [g32P]ATP, and the reaction products containing PtdIns [32P]3-phosphate are separated by thin layer chromatography. The labeled reaction products are quantitated by a Molecular Dynamics Storm System.
       

  5. GTPase activation measurements:

    1. Ras: Induction of Ras will be directly measured by labeling cells metabolically with [32P]-inorganic phosphate, or by permeabilization and addition of [g32P]GTP. Ras will be immunoprecipitated and Ras-bound [32P]-labeled guanine nucleotides will be analyzed and quantitated after separation by polyethyleneimine-coated thin layer chromatography plates. Personnel within the core facility have experience with this assay (10).

    2. Rho-family: Rac, Rho and CDC42 have been assessed by membrane association of the proteins (20), detected by immunoblot; or by GTP/GDP ratios obtained from metabolically-labeled cells (21). Our personnel have experience using both strategies, and these will be applied to experimental systems on an ad hoc basis. After immunoprecipitation, [32P]-labeled guanine nucleotides bound to Rho-family GTPases will be fractionated by ion-exchange TLC, as above. Positions of the [32P]-labeled guanine nucleotides will be determined according to the mobility of the unlabeled GDP and GTP commercial standards.
       

  6. Transient transfections:

    Transient and stable transfection of hematopoietic cells is a rapid and informative method of determining the relationship between enzymes in a signaling pathway. Transfections of lymphocytes with a limiting amount of a reporter enzyme and co-transfection with a relatively large amount of a dominant-positive or -negative signaling enzyme mutant has been a successful strategy to explore a potential relationship between two enzymes. The core facility maintains several cultured lymhocytic and myelocytic cell line models, and has developed and optimized techniques for their transfection. All of this information and technology is available to OMRF investigators.
     

  7. References:

    1. DeMagistris, M.T., J.Alexander, M. Coggeshall, A. Altman, F.C. Gaeta, H.M. Grey, and A. Sette. 1992. Antigen analog-major histocompatibility complexes act as antogonists of the T cell receptor. Cell 68:625.

    2. Pradhan, M., and K.M. Coggeshall. 1997. Activation-induced bi-dentate SHIP and Shc interaction in B lymphocytes. J. Cell. Biochem. 67:32.

    3. Phee, H., A. Jacob, and K.M. Coggeshall. 2000. Enzymatic activity of the Src Homology 2 domain-counting inositol hosphatase is regulated by a plasma membrane location. J. Biol. Chem. 275:19090.

    4. Songyang, Z. 1999. Recognition and regulation of primary-sequence motifs by signaling modular domains. Prog. Biophys. Mol. Biol. 71:369.

    5. Songyang, Z., S.E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W.G. Haser, F. King, T. Roberts, S. Ratnofsky, R.J. Lechleider, and et al. 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72:767.

    6. Yu, H., J.K. chen, S. Feng, D.C. Dalgarno, A.W. Brauer, and S.L. Schreiber. 1994. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell 76:933.

    7. Dalgarno, D.C., M.C. Botfield, and R.J. Rickles. 1997. SH3 domains and drug design: ligands, structure, and biological function. Biopolymers 43:383.

    8. Tridandapani, S., T.Kelley, M. Pradhan, D. Cooney, L.B. Justement, and K.M. Coggeshall. 1997. Recruitment and phosphorylation of SHIP and Shc to the B cell Fcgamma ITIM peptide motif. Mol. Cell. Biol. 17:4305.

    9. Tridandapani, S., M. Pradhan, J.R. LaDine, S. Garber, C.L. Anderson, and K.M Coggeshall. 1999. Protein interactions of Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP): association with Shc displaces SHIP from FCgamaRIIb in B cells. J. Immunol. 162:1408.

    10. Tridandapani, S., G.W. Chacko, J.R. v. Brocklyn, and K.M. Coggeshall. 1997. Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions. J. Immunol. 158:1125.

    11. Chacko, G.W., S. Tridandapani, J. Damen, L. Liu, G. Krystal, and K.M. Coggeshall. 1996. Negative signaling in B-lymphocytes induces tyrosine phosphorylation of the 145 kDa inositol polyphosphate 5-phosphatase, SHIP. J. Immunol. 157:2234.

    12. Boyle, W.J., P. van der Geer, and T. Hunter. 1991. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional speartion on thin-layer cellulose plates. Meth. Enz. 201.

    13. Tridandapani, S., H. Phee, L. Shivakumar, T. Kelley, and K.M. Coggeshall. 1999. Role of SHIP in FcgammaRIIb-mediated inhibition of Ras activation in B cells. Mol. Immunol. 35:1135.

    14. Chen, Z.Z., K.M. Coggeshall, and J.C. Cambier. 1986. Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes. J. Immunol. 136:2300.

    15. Baier, G., D. Telford, L. Giampa, K.M. Coggeshall, G. Baier-Bitterlich, N. Isakov, and A. Altman. 1993. Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly i hematopoietic cells. J. Biol. Chem. 268:4997.

    16. Jacob, A., D. Cooney, S. Tridandapani, T. Kelley, and K.M. coggeshall. 1999. FcgammaRIIb modulation of surface immunoglobulin-induced Akt activation in murine B cells. J. Biol. Chem. 274:13704.

    17. Chacko, G.W., A.M. Duchemin, K.M. Coggeshall, J.M. Osborne, J.T. Brandt, and C.L. Anderson. 1994. Clustering of the platelet Fc gamma receptor induces noncovalent association with the tyrosine kinase p72syk. J. Biol. Chem. 269:32435.

    18. Sarkar, S., K. Schlottmann, D. Cooney, and K.M. Coggeshall. 1996. negative signaling via FcgammaIIb1 in B cells blocks phospholipase Cgamma2 phosphorylation but not Syk or Lyn activation. J. Biol. Chem. 271:20182.

    19. Chacko, G.W., J.T. Brandt, K.M. Coggeshall, and C.L. Anderson. 1996. Phosphoinositide 3-kinase and p72syk noncovalently associate with the low affinity Fc gamma receptor on human platelets through an immunoreceptor tyrosine-based activation motif. Reconstitution with synthetic phosphopeptides. J. Biol. Chem. 271:10775.

    20. Bokoch, G.M., B.P. Bohl, and T.H. Chuang. 1994. guanine nucleotide exchange regulates membrane translocation of Rac/Rho GTP-binding proteins. J. Biol. Chem. 269:31674.

    21. Gulbins, E., K.M. Coggeshall, B. Brenner, K. Schlottmann, O. Linderkamp, and F. Lang. 1996. Fas-induced apoptosis is mediated by activation of a Ras and Rac protein-regulated signaling pathway. J. Biol. Chem. 271:26389.