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Purification and characterization of full-length monomeric TEC family kinase, ITK

  1. Author:
    Rathnayake, Udumbara M
    Wada, Junya
    Wall, Vanessa E
    Jones,Jane
    Jenkins, Lisa M
    Andreotti, Amy H
    Samelson, Lawrence E

  2. Author Address

    Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA., Protein Expression Laboratory and RAS Reagents Core, Frederick National Laboratory for Cancer Research, Frederick, MD 21702 USA., Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA., Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50014 USA., Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA. Electronic address: samelsonl@mail.nih.gov.,
    1. Year: 2025
    2. Date: Jan 31
    3. Epub Date: 2025 01 31
  2. Journal: Protein Expression and Purification
    1. 229
    2. Pages: 106682
  4. Type of Article: Article
  5. Article Number: 106682
  1. Abstract:

    An early step in the activation of T cells via the T cell antigen receptor is the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1) by the TEC family tyrosine kinase, interleukin-2 (IL-2) inducible T cell kinase (ITK). PLC-gamma1 activation occurs within a multi-protein complex comprised of the enzymes ITK, PLC-gamma1, and VAV, and the adapter molecules, LAT, Gads, SLP-76, and NCK. Studies of ITK activation and the role of this heptameric complex in regulating ITK activation and function have not been possible due to the lack of success in the expression and purification of full-length, monomeric ITK protein. In this study, we have produced soluble full-length wild-type ITK protein by co-expressing an N-terminal solubility-tagged ITK construct with a kinase-specific co-chaperone CDC37 in an insect cell line. Although the majority of the purified ITK protein is oligomerized, there is a 13-fold increase in the yield of monomeric protein production compared to the last reported purification. Previous studies suggest that the ITK oligomerization is mediated by intermolecular interactions. We created several mutants to disrupt these self-associations. Expression of one of these, the C96E/T110I mutant, produced 20 times more monomer than the wild-type construct. The in vitro characterization of these protein constructs showed that the purified protein is stable and functional. This successful purification and in vitro characterization of full-length monomeric ITK protein will aid in understanding the mechanism by which ITK is recruited into the heptameric complex and is enabled to phosphorylate and activate PLC-gamma1. Copyright © 2025. Published by Elsevier Inc.

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  2. DOI: 10.1016/j.pep.2025.106682
  3. PMID: 39894064
  4. PII : S1046-5928(25)00024-5

Library Notes

  1. Fiscal Year: FY2024-2025
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