Elucidation of regulatory mechanisms of the ITK and its substrate PLCγ1

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Devkota, Sujan
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Amy H. Andreotti
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Biochemistry, Biophysics and Molecular Biology

The Department of Biochemistry, Biophysics, and Molecular Biology was founded to give students an understanding of life principles through the understanding of chemical and physical principles. Among these principles are frontiers of biotechnology such as metabolic networking, the structure of hormones and proteins, genomics, and the like.

The Department of Biochemistry and Biophysics was founded in 1959, and was administered by the College of Sciences and Humanities (later, College of Liberal Arts & Sciences). In 1979 it became co-administered by the Department of Agriculture (later, College of Agriculture and Life Sciences). In 1998 its name changed to the Department of Biochemistry, Biophysics, and Molecular Biology.

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  • Department of Biochemistry and Biophysics (1959–1998)

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This dissertation studies the regulatory mechanism of IL-2 inducible tyrosine kinase (ITK) and its substrate phospholipase C γ1 (PLCγ1). ITK and PLCγ1 are key mediators of the signaling pathway downstream of the T cell receptor that results in the T cell part of the adaptive immune response. In T cells, ITK is phosphorylated and activated by LCK. ITK then phosphorylates PLCγ1 at tyrosine 783 and activates phospholipase activity. This phosphorylation is dependent upon various coordinated intermolecular and intramolecular interactions within and between ITK and PLCγ1. This dissertation identifies and characterizes various regulatory interactions in ITK and its substrate PLCγ1 that regulates ITK mediated phosphorylation of PLCγ1.

For ITK, this thesis explores the function of the N-terminal Pleckstrin homology (PH) domain in regulating the activation of the ITK. The ITK PH domain engages in direct interaction with ITK kinase domain and the region of the kinase domain interaction is mapped in the ITK PH domain. Mutations in ITK PH domain, that disrupts its interaction with kinase domain, lead to the increase in the activity of ITK. The ITK interaction surface mapped on the ITK PH domain lies adjacent to its phosphatidylinositol (3,4,5)-triphosphate (PI (3,4,5) P3) binding pocket. Hence, IP4, the soluble head group of (PI (3,4,5) P3) competes with the ITK kinase domain for ITK PH domain binding. Also, PI (3,4,5) P3 binding of ITK PH domain increases the catalytic activity of the ITK. In addition, PI (3,4,5) P3 binding increases the activation loop Y511 accessibility for LCK phosphorylation. This study expanded our knowledge on regulation of ITK by its N terminal PH domain (Chapter 3).

ITK is a key modulator of immune response and has therefore been very attractive target for small molecule intervention for immunity related diseases such as autoimmune disease and asthma. The key novel regulatory ITK PH/kinase site can be a plausible allosteric target for small molecule discovery efforts. Part of this thesis contributes to assay development to detect the ITK PH/Kinase interaction in cells. Bimolecular Fluorescence complementation (BiFC) assay confirms that the ITK PH/Kinase interaction occurs in cell (Chapter 4). This assay can now be used to screen for small molecules that modulate the ITK PH/Kinase interaction.

For PLCγ1, this thesis studies the mechanism of disruption of its autoinhibitory conformation. The PLCγ1 autoinhibitory conformation is characterized by an intramolecular interaction between the C terminal SRC homology 2 (SH2C) domain and the adjacent linker containing Y783. This conformation makes the crucial phosphorylation target (Y783) inaccessible to ITK. I have described the mechanism on how this autoinhibitory interaction is broken (Chapter 2). Our results suggest that the scaffold protein SLP-76 disrupts the autoinhibitory PLCγ1 conformation. More specifically, SLP-76 phosphotyrosine 173 (Y173) binds the SH2C domain of the PLCγ1, competing with the autoinhibitory conformation of PLCγ1 and releasing the linker, making the Y783 more accessible to ITK. Our results identify the new role of the scaffold protein SLP-76 adding to its previously described role in co-localizing the enzyme and substrate pair. This role is defined as the substrate priming as SLP-76 primes PLCγ1, the substrate for ITK for efficient phosphorylation. Our results provide further understanding of the regulation of ITK mediated phosphorylation of PLCγ1 and create a foundation for small molecule discovery efforts that should yield new ways to either enhance or diminish T cell function.

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Sun Jan 01 00:00:00 UTC 2017