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Research Experience in Molecular Biotechnology & Genomics Summer 2007 Center for Integrated Animal Genomics Study of the Role of Asp 181 in Itk Kinase Autophosphorylation through Mutation Tasida Fisher1, Raji Joseph2, Eli Mussleman2, and Amy Andreotti2 1Iowa Lakes Community College; 2Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University Method & Materials •Mutations were introduced into a template SH3-SH2 plasmid using QuikChange® Site Directed Mutagenesis Kit from Stratagene®. •The mutated plasmids were multiplied in-vivo by transforming XL1-Blue bacteria. •These transformed bacteria were cultured, then lysed to extract the plasmids. •Plasmids were transformed into BL-21 bacteria that synthesized the mutated protein in-vivo. •These bacteria were lysed, and protein was extracted using nickel columns (Figure 5). •The protein was purified and concentrated to 1 mM. Figure 1: Proposed computer model of Itk kinase SH3-SH2 domain complex based on the crystalline structures of activated Itk kinase and the SH3-SH2 domains. The Itk Kinase domain is highlighted in blue, the SH3 domain in gray, the SH2 domain in red, the amino acid residues involved in the docking mechanism within the SH2 domain in yellow, and Tyr 180 and Asp 181 within the SH3 domain in orange and green respectively. The insert illustrates the probable position of Tyr 180 and Asp 181 within the active site of Itk kinase. Structure generated using PyMOL. Abstract The role of Aspartic Acid 181 in the autophosphorylation of Tyrosine 180 in the Src homology-3 (SH3) domain of Interleukin-2 kinase (Itk) is characterized by observing the phosphorylation efficiencies of SH3-SH2 substrates mutated at Asp 181. While mutation at this site did not destroy the substrate’s ability to be phosphorylated by Itk, differences in phosphorylation efficiencies were observed among different mutations. Introduction Itk, involved in signal transduction in T-cells, phosphorylates two substrates: Tyr 783 on phospholipase C1, and Tyr 180 within its own SH3 domain. Itk has been shown to recognize its substrates by a remote docking mechanism involving the SH2 domain. The purpose of this experiment is to determine whether Asp 181, adjacent to Tyr 180 has a similar role in preserving the substrate specificity of Itk. (figure 1) Figure 2: Diagram of amino acids under study. Created with CS ChemDraw StdTM. Substrates consisting of the SH3 and SH2 domains of Itk were created. Asp 181 within this substrate was mutated to either alanine, lysine, or serine (figures 2, 3, & 4). The ability of each mutant substrate to be phosphorylated by Itk kinase was measured using an in-vitro kinase assay followed by western blotting with an anti-phospho-tyrosine specific antibody and chemiluminescent development. •Each mutated protein was allowed to react with full-length Itk enzyme and ATP in an in-vitro kinase assay buffer for one hour. •The samples were boiled, separated by SDS-PAGE followed by western blotting with an anti-phospho-tyrosine specific primary antibody and chemiluminescent development. Figure 5: SDS PAGE Gel showing different stages of nickel column purification of ITK SH3* SH2* (D181A) ~ 24 kDa. Lane 1, Low Range Standards molecular ladder. Lane 2, total lysate. Lane 3, supernatant. Lane 4, flow through. Lane 5, wash buffer. Lane 6, second elution. •The western blot was exposed to x-ray film to measure the presence of phosphorylated substrates. Figure 6: X-ray exposure showing the phosphorylation levels of the mutant proteins. The negative control consists of a wild-type sample that did not have Itk added to it during the kinase assay. Figure 7: SDS PAGE gel showing the actual level of protein in each sample in the kinase assay. The lane containing the D181K mutant protein indicates that there may be less protein available to be phosphorylated, which may explain the lighter corresponding band in the X-ray exposure (figure 6). Results & Discussion •Mutation to alanine does not significantly reduce phosphorylation (figure 6). •Mutation to lysine or serine appears to reduce phosphorylation. However, the reduced phosphorylation of D181K may be caused by a lower concentration of protein (figures 6, 7). •Since some mutation at Asp 181 in Itk seems to be tolerated, Asp 181 probably does not play a direct role in conserving the specificity of Itk to its SH3 domain. •Some mutations at Asp 181 appear to structurally inhibit the phosphorylation of Tyr 180. For example, replacing the small, negatively charged amino acid of aspartic acid with the large, positively charged side chain of lysine may block the access of Tyr 180 to the active site of Itk kinase. •These results are highly preliminary. More replications of this experiment need to be conducted. A radioactive assay is also needed to determine more precisely the level of phosphorylation in each mutant protein, and structural studies need to be conducted to confirm that these mutations do not cause decreased phosphorylation levels by denaturing the protein. Figure 3: Domain structure of full-length Itk, which was used as the enzyme to phosphorylate the substrates under study. •A study of a wider variety of mutations at Asp 181 and structural analysis of the entire Itk kinase SH3-SH2 complex can further characterize the function of Asp 181 in Itk. Acknowledgments Figure 4: Domain structure of the substrate. It consists of the Itk SH3 domain, which contains a tyrosine autophosphorylated by Itk, and the Itk SH2 domain, which contains the remote docking area needed to effectively react with Itk kinase. The amino acid sequence of the SH3 domain was expanded to highlight the site of autophosphorylation, Tyr 180 (boxed in black), and to highlight the amino acid mutated in this study, Asp 181 (boxed in red). Figures 2 & 3 were adapted with permission from a poster presented in 2005 by Eli Mussleman. 1. 2. 3. 4. 5. I would like to thank the Andreotti lab for their patience and hospitality this summer: Lie Min, Andrew Severin, and Ruo Xu. I can hardly imagine a better group of people to work with. I would especially like to thank Eli Mussleman, Raji Joseph, and Amy Andreotti for the knowledge and guidance that made this project come together. I would also like to thank the faculty and staff at Iowa Lakes Community College that made my internship here possible: Matt Abbott, Gary Phillips, Jolene Rogers, and Robert Klepper. Also, thank you to the NSF and to Max Rothschild for coordinating the REU Program. References Bose, R, Holbert, M. A., Pickin, K. A., and Cole, P. A. (2006) Protein tyrosine kinase-substrate interactions. Current Opinion in Structural Biology. 16, 668-675. Pere-Villar, J. J., and Kanner, S. B. (1999) Regulated association between the tyrosine kinase Emt/Itk/Tsk and phospholipase-C 1 in human T lymphocytes. J. Immunol. 163, 6435-6441. Wilcox, H. M., and Berg, L. J. (2003) Itk phosphorylation sites are required for functional activity in primary T cells, J. Biol. Chem. 278, 37112-37121. Nore, B. f., Mattsson, P. T., Antonsson, P., Backesjo, C. M., Westlund, A., Lennartsson, J., Hoansson, H., Low, P., Ronnstrand, L., and Smith, C. I. (2003) Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases, Biochim. Biophys. Acta 1645, 123-132. Joseph, Raji E., Min, L., Xu, R., Musselman, E. D., and Andriotti, A. H. (2007) A Remote Substrate Docking Mechanism for the Tec Family Tyrosine Kinases, Biochemistry, 46, 5595-5603. Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371