Molecular Regulation and Function of the Protein Tyrosine Kinase Pyk2 in Cytotoxic T Lymphocytes

  • Author / Creator
    Cheung, Samuel MS
  • Cytotoxic T lymphocytes (CTL) play a critical role in immune surveillance and elimination of virus-infected and malignant cells. CTL utilize two major cell-contact dependent mechanisms to kill antigen-bearing target cells. One mechanism involves the directional release of cytolytic molecules stored in granules. Another mechanism is through surface expression of Fas ligand (FasL), which bind Fas-expressing target cells to induce apoptosis. My research objectives are to dissect some of the molecular events and mechanisms that CTL employ to destroy infected and/or tumor cells, with a particular focus on elucidating the contribution of the proline-rich tyrosine kinase (Pyk2) to CTL-mediated cytotoxicity. Pyk2 was initially identified as a calcium-dependent kinase but how Pyk2 activation is regulated by calcium in T cells had not been addressed. I found that calcium-mediated Pyk2 activation is regulated by reactive oxygen species (ROS). CTL stimulated with reagents that increase intracellular calcium concentration trigger the production of hydrogen peroxide (H2O2). Stimulation of CTL with H2O2 elicits Pyk2 activation and calcium-induced Pyk2 activation requires Erk and Src-family kinase. My data indicate that H2O2 also regulates Erk, which likely acts on Src-family kinase to mediate Pyk2 activity. In my study of CTL function, I found that Pyk2 regulates CTL migration on ICAM-1. Inhibition or knockdown of Pyk2 results in defective CTL de-adhesion from an ICAM-1 coated surface. Deregulation of Pyk2 leads to a significant reduction in overall CTL motility. My data indicate that Pyk2 is recruited to the site of integrin activation near the membrane and that Pyk2 phosphorylated on different tyrosines have distinct cellular localization in CTL. Inhibition of Pyk2 impairs intracellular LFA-1 distribution suggesting that Pyk2 regulates LFA-1 recycling. My results indicate that inhibition of Pyk2 impairs CTL degranulation. Upon CTL engagement of the target cell, there is reorientation of the microtubule organizing center (MTOC) towards the target cell which is thought to allow for directional release of the granules. Pyk2 contributes to MTOC reorientation in CTL towards the target cell. Taken together, my data show that Pyk2 regulates multiple aspects of CTL adhesion and function.

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    Doctor of Philosophy
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