Non-conventional Regulation of Itk and Akt Kinases in T Cells and Dendritic Cells
Date of Award
Doctor of Philosophy (PhD)
Pleckstrin homology (PH) domains are abundantly found in signaling proteins that perform diverse cellular functions, including signal transductions pathways, cell cytoskeleton rearrangements, vesicular transportation and cell cycle control. The PH domain-containing kinases, Itk and Akt are important for immune cell development, differentiation and function. Itk and Akt activation is dependent on binding between their PH domains and the membrane lipid PtdIns(3,4,5)P3 (PIP3). However, we have identified non-conventional ligands for the Itk and Akt PH domain that regulate the magnitude and kinetics of Itk and Akt activity. We found that the intracellular Ca2+ sensing chaperone protein, Calmodulin (CaM) regulates Itk activity by directly interacting with the Itk PH domain in T cells. In collaboration with Dr. Amy Andreotti's lab, we mapped the interaction between CaM and the Itk PH domain using nuclear magnetic resonance (NMR) spectroscopy. We found that Ca2+/CaM and PIP3 cooperated to enhance binding to either ligand. Disruption of Ca2+/CaM binding attenuated Itk recruitment to the membrane and subsequent activation of PLCγ1. This suggests that Ca2+/CaM binding to the Itk PH domain acts in a positive feedback loop to potentiate and sustain Ca2+ signaling. Disruption of the feedback loop abrogated Itk-dependent production of the pro-inflammatory cytokine IL-17A in CD4+ T cells.
Inositol 1,3,4,5-tetrakisphosphate (IP4) is a soluble second messenger that is generated following stimulation of ITAM-dependent surface receptors. IP4 bears considerable structural similarity to the ligand-binding head group of PIP3 and has been shown to co-regulate PIP3-binding PH domains. IP4 is critical for T cell, B cell and NK cell development and activation. However, whether IP4 has a similar regulatory role in dendritic cell (DC) function is not yet known. Here, we found that IP4 regulates the migratory potential of dendritic cells. Endogenous migratory DCs from IP4 deficient mice and bone marrow derived dendritic cells (BMDCs) migrated more efficiently to peripheral lymph nodes in vivo. IP4 deficient BMDCs also have enhanced secretion of pro-inflammatory cytokines/chemokines. Enhanced dendritic cell function in IP4 deficient mice correlated with increased Akt activity, which in turn dampens ERK signaling. This suggests that normally, IP4 supports ERK activation by negatively regulating Akt and its down-stream signals. Together, our data support an important role for IP4 in DC migration and cytokine secretion, identifying IP4 as a dual regulator of Akt and ERK signaling pathways that controls DC function.
Chair and Committee
Yina H Huang
Paul M Allen, Gaya K Amarasinghe, Kenneth M Murphy, Andrey S Shaw, Wojciech A Swat
Wang, Xinxin, "Non-conventional Regulation of Itk and Akt Kinases in T Cells and Dendritic Cells" (2014). Arts & Sciences Electronic Theses and Dissertations. 345.