David Mitchell, Ph.D.
Assistant Professor, College Of Medicine Molecular Medicine
College Of Medicine Molecular Medicine
Assistant Professor, Dept. of Molecular Medicine, College of Medicine, University of South Florida
12901 Bruce B. Downs Blvd.
Tampa, FL 33612
Academic Email: email@example.com
Academic Phone: (813) 974-2946
- Ph.D., Biochemistry, University of Iowa, 1996
- PHD, Biochemistry, University Of Iowa, 1995
- B.Sc., Genetics, The Ohio State University, 1985
- The primary goal of my research is to address and understand some of the complex molecular signal transduction mechanisms in nature using the model organism, Saccharomyces cerevisiae. At present I am focusing on two research objectives: a) understanding the complex interplay between cellular membranes and proteins that are peripherally associated with these membranes and b) characterizing the factors which regulate Palmitoyl Acyl Transferases (PAT), an enzyme that posttranslationally attaches long chain fatty acids to proteins.
In order to study the dynamics of proteins along cellular membranes, I have constructed a genetically tractable system to evaluate the movement of Ras2 in relation to the inner plasma membrane of the yeast cell. This system utilizes Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) of live yeast cells to monitor the diffusion and membrane exchange of GFP-tagged Ras2 molecules at the plasma membrane. Ras2 begins as a cytosolic protein, then immediately becomes farnesylated at its C-terminus resulting in membrane localization. In addition to farnesylation, Ras2 also undergoes palmitoylation while membrane localized as well as being methylesterified. Through genetic manipulations, we will be able to assess the contributions each and all of these posttranslational modifications make to the overall association of Ras2 to the plasma membrane.
In addition, I am also constructing High Throughput Screening platforms to identify modulators of the Ras Palmitoyl Acyl Transferases (RasPAT) with the goal of identifying inhibitors of Ras palmitoylation. Inhibitors of Ras function are excellent candidates for the development of cancer chemotherapeutic drugs, given the central role of Ras signaling in most cancer cells. Ras PAT carries out the posttranslational addition of palmitate to the C-terminus of Ras proteins. This lipophilic addition is required for subcellular trafficking and membrane assembly of active Ras signaling complexes. Since the discovery of Ef2/Erf4 (yeast RasPAT) by Robert Deschenes and colleagues, the palmitoylation field has grown exponentially. However, the identification of tools to study these proteins has severely lagged in comparison. Identification of inhibitors and modulators will be a substantial move forward in cancer biology.
- American Society of Cell Biology (Member, 2007 - Present)
- Genetics Society of America (Member, 1996 - 2001)
- American Society for Biochemistry and Molecular Biology (Member, 1991 - 1995)
- Gayatri Mitchell, Kayoko Ishizuka, Robert J. Deschenes and David A. Mitchell Saccharomyces cerevisiae Erf4 protects Erf2 from C-terminal ubiquitinylation without participating in the enzymatic mechanism. Manuscript in Preparation. , 2011.
- Ming Hu, Feming Wang, Xin Li, Christopher Q. Rogers, Xiaomei Liang, Brian N. Finck, Mayurranjan Mitra, Ray Zhang, David A. Mitchell and Min You Regulation of Hepatic Lipin-1 by Ethanol: Role of AMPK-SREBP-1 Signaling. Hepatology (In Press). , 2011.
- Kalyan C. Vinnakota, Daniel A. Beard, Robert J. Deschenes and David A. Mitchell. The yeast Ras2 C-terminal posttranslational modifications act in concert to regulate plasma membrane affinity and diffusivity. Manuscript in preparation.. , 2011.
- Kalyan C. Vinnakota, David A. Mitchell, Daniel A. Beard, Robert J. Deschenes and Tetsuro Wakatsuki. Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching. Phys Biol. 7(2) : 26011, 2010.
- David A. Mitchell, Gayatri Mitchell, Yiping Ling, Cheryl Budde and Robert J. Deschenes Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes. J Biol Chem. 285(49) : 38104, 2010.
- Phalgun P. Joshi, Martin Hirst, Tom Malcolm, Jennifer Parent, David A. Mitchell, Karen Lund and Ivan Sadowski. Identification of protein interaction antagonists using the Repressed Trans-activator two-hybrid system. Biotechniques. 5(42) : 635-644, 2007.
- Vottero, E.Mitchell, DA.Page, MJ.MacGillivray, RT.Sadowski, IJ.Roberge, M.Mauk, AG. Cytochrome b(5) is a major reductant in vivo of human indoleamine 2,3-dioxygenase expressed in yeast. FEBS letters. 580(9) : 2265-8, 2006. http://www.ncbi.nlm.nih.gov/pubmed/16574111
- Mitchell, DA.Vasudevan, A.Linder, ME.Deschenes, RJ. Protein palmitoylation by a family of DHHC protein S-acyltransferases. Journal of lipid research. 47(6) : 1118-27, 2006. http://www.ncbi.nlm.nih.gov/pubmed/16582420
- Sadowski, I.Mitchell, DA. TFII-I and USF (RBF-2) regulate Ras/MAPK-responsive HIV-1 transcription in T cells. European journal of cancer (Oxford, England : 1990). 41(16) : 2528-36, 2005. http://www.ncbi.nlm.nih.gov/pubmed/16223582
- Goehring, AS.Mitchell, DA.Tong, AH.Keniry, ME.Boone, C.Sprague, GF. Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome activation. Molecular biology of the cell. 14(4) : 1501-16, 2003. http://www.ncbi.nlm.nih.gov/pubmed/12686605
- Dong, X.Mitchell, DA.Lobo, S.Zhao, L.Bartels, DJ.Deschenes, RJ. Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae. Molecular and cellular biology. 23(18) : 6574-84, 2003. http://www.ncbi.nlm.nih.gov/pubmed/12944483
- Mitchell, DA.Sprague, GF. The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/M transition in Saccharomyces cerevisiae. Molecular and cellular biology. 21(2) : 488-500, 2001. http://www.ncbi.nlm.nih.gov/pubmed/11134337
- Bartels, DJ.Mitchell, DA.Dong, X.Deschenes, RJ. Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae. Molecular and cellular biology. 19(10) : 6775-87, 1999. http://www.ncbi.nlm.nih.gov/pubmed/10490616
- Naqvi, SN.Zahn, R.Mitchell, DA.Stevenson, BJ.Munn, AL. The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Current biology : CB. 8(17) : 959-62, 1998. http://www.ncbi.nlm.nih.gov/pubmed/9742397
- Mitchell, DA.Deschenes, RJ. Characterization of protein prenylation in Saccharomyces cerevisiae. Methods in enzymology. 250: 68-78, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7651186
- Farh, L.Mitchell, DA.Deschenes, RJ. Farnesylation and proteolysis are sequential, but distinct steps in the CaaX box modification pathway. Archives of biochemistry and biophysics. 318(1) : 113-21, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7726551
- Mitchell, DA.Farh, L.Marshall, TK.Deschenes, RJ. A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae. The Journal of biological chemistry. 269(34) : 21540-6, 1994. http://www.ncbi.nlm.nih.gov/pubmed/8063791
- Mitchell, DA.Marshall, TK.Deschenes, RJ. Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast. Yeast (Chichester, England). 9(7) : 715-22, 1993. http://www.ncbi.nlm.nih.gov/pubmed/8368005
- Swierczynski, J.Mitchell, DA.Reinhold, DS.Salati, LM.Stapleton, SR.Klautky, SA.Struve, AE.Goodridge, AG. Triiodothyronine-induced accumulations of malic enzyme, fatty acid synthase, acetyl-coenzyme A carboxylase, and their mRNAs are blocked by protein kinase inhibitors. Transcription is the affected step. The Journal of biological chemistry. 266(26) : 17459-66, 1991. http://www.ncbi.nlm.nih.gov/pubmed/1680129
- Stapleton, SR.Mitchell, DA.Salati, LM.Goodridge, AG. Triiodothyronine stimulates transcription of the fatty acid synthase gene in chick embryo hepatocytes in culture. Insulin and insulin-like growth factor amplify that effect. The Journal of biological chemistry. 265(30) : 18442-6, 1990. http://www.ncbi.nlm.nih.gov/pubmed/2170411
- Ma, XJ.Salati, LM.Ash, SE.Mitchell, DA.Klautky, SA.Fantozzi, DA.Goodridge, AG. Nutritional regulation and tissue-specific expression of the malic enzyme gene in the chicken. Transcriptional control and chromatin structure. The Journal of biological chemistry. 265(30) : 18435-41, 1990. http://www.ncbi.nlm.nih.gov/pubmed/2211712
- Alan G. Goodridge, James F. Crish, Dominic A. Fantozzi, Manuel J. Glynias, F.Bradley Hillgartner, Stephen A. Klautky, Xiao-Jun Ma, David A. Mitchell and Lisa M. Salati. Regulation of Avian Genes Involved in Fatty Acid Synthesis. Journal of Animal Science. 66: 49-66, 1989.
- Research Assistant Professor Medical College of Wisconsin 2007 - 2008)
- Research Scientist II Medical College of Wisconsin 2005 - 2008)
- Research Associate Faculty (Non-Tenure) University of British Columbia 2003 - 2005)
- Project Leader/Senior Scientist Interomex BioPharmaceuticals, Inc. 2001 - 2003)
- Postdoctoral Fellow (Institute of Molecular Biology, University of Oregon 1996 - 2001)
- Subrumanian Thesis Award 1996)