Transcript Document

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 C1, 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.
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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