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Natalie G. AHN
Office Phone: 303 492 4799
E-mail: Natalie.Ahn@colorado.edu
FAX: 303 492 2439
Lab: Cristol Chemistry 200, 215
Lab Phone: 303 492 7794, 303 492 7769, 303 735 4915
Group Website:
Ph.D.: University of California, Berkeley, 1985
Postdoctoral Fellow: University of Washington,
Signal Transduction and Protein Phosphorylation
A major goal of our research is to understand how phosphorylation controls cell signal transduction, by identifying protein kinases and phosphatases that are controlled by growth factors and examining their mechanisms of regulation. A second goal is to develop new techniques for analyzing post-translational modifications of proteins involved in signaling.
An intracellular signal transduction pathway, called the MAP kinase cascade, is rapidly stimulated in response to growth factors. Enzymes in this pathway include pp90 ribosomal S6 kinase, mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MKK, also referred to as MEK), which form three tiers of a protein kinase cascade in which pp90rsk is phosphorylated and activated by MAPK, and MAPK is phosphorylated and activated by MPKK. MPKK is phosphorylated and activated by any of three protein kinases, Raf-1, MEK kinase, and Mos, and thus is a convergence point for diverse signaling pathways triggered upon cell surface receptor activation. Several transcription factors are downstream targets for these kinases, thus, the MAP kinase cascade is a key pathway allowing transcriptional regulation by extracellular stimuli.
Our aim is to understand the regulation of MKK and its role in cell growth and differentiation. We have identified several phosphorylation sites on this enzyme, and are examining their contributions to kinase activation. Mutants of MKK that are either constitutively active or catalytically inactive were designed, which, upon transfection into cultured mammalian cells respectively, enhance or block signal transduction through the MAP kinase pathway. Overexpression of constitutively active MKK mutants led to transformation in several types of cells. Interestingly, cell types respond instead by cell cycle arrest and differentiation. We are currently examining downstream cellular targets of MAP kinase and MKK to explain how such different cellular responses can be controlled by the same pathway.
Electrospray ionization mass spectrometry (ESI-MS) enables determination of protein or nucleic acid masses to uncertainties of 1 in 10,000 Da. Experimental approaches we use in our laboratory include (i) liquid chromatography coupled to mass spectrometry (LC/MS), which separates molecules by high performance liquid chromatography followed by mass determination, (ii) tandem mass spectrometry (MS/MS, LC/MS/MS), which fragments ions by collision-induced dissociation, allowing peptide and nucleic acid sequences and specific sites of covalent modification to be determined, and (iii) deuterium exchange coupled to mass spectrometry, which measures solvent accessibility of protein backbone amides.
LC/MS and LC/MS/MS are being used to identify phosphorylation sites and autophosphorylation sites on MKK, and other proteins. Deuterium exchange experiments are being combined with site directed mutagenesis to identify conformational differences between inactive vs. active forms of MKK and MAPK. We are also using mass spectrometry to examine masses of components within large protein/nucleic acid complexes with the aim of identifying novel protein post-translational modifications under growth factor control.
Selected Publications
Resing, K.A., Meyer-Arendt, K., Mendoza, A.M., Aveline-Wolf, L.D., Jonscher, K.R., Pierce, K.G., Old, W.M., Cheung, H.T., Russell, S., Wattawa, J.L., Goehle, G.R., Knight, R.D., Ahn, N.G. (2004) Improving reproducibility and sensitivity in identifying human proteins by shotgun proteomics. Analytical Chemistry (revision submitted).
Bernard, K., Litman, E., Fitzpatrick, J.L., Shellman, Y.G., Argast, G., Polvinen, K., Everett, A.D., Fukasawa, K, Norris, D.A., Ahn, N.G., Resing, K.A. (2003) Functional proteomic analysis of melanoma progression. Cancer Research. 63:6716-6725.
Haydon, C.E., Eyers, P.A., Wolf, L.S., Resing, K.A., Maller, J.L. and Ahn, N.G. (2003) Identification of novel phosphorylation sites on Xenopus laevis Aurora A using improved IMAC methodology. Mol. Cell. Proteomics. 2:1055-1067.
Galasinski S., Louie D.F., Gloor, K.K., Resing, K.A., and Ahn, N.G. (2002) Global regulation of post-translational modifications on core histones. J. Biol. Chem. 277:2579-88.
Ahn, N.G. and Resing, K.A. (2001) Towards the phosphoproteome (News and Views). Nature Biotechnol. 19:317-318.
Lewis, T.S., Hunt, J.B., Aveline, L.D., Jonscher, K.R., Louie D.F., Yeh J.M., Nahreini, T.S., Resing, K.A., Ahn, N.G. (2001) Identification of Novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Molecular Cell. 6:1-20.
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