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Exploring the Influence of PKC-theta Phosphorylation on Notch1 Activation and T Helper Cell Differentiation

The T cell-specific kinase, Protein Kinase C theta (PKCq) is essential to T cell activation and differentiation. PKCq integrates T cell receptor (TCR) and CD28 signaling, and ultimately activates transcription factors necessary for full T cell activation, proliferation, survival, and differentiation into T helper (Th) subsets. Th1, Th2, Th17 and Treg cells compose the four major lineages of T helper cells, differentiated from CD4 T cells, and each have different requirements for PKCq. PKCq, itself, is regulated through phosphorylation of specific resides, including tyrosine (Y)90 and threonine (T)538. Following T cell stimulation, PKCq is phosphorylated on Y90 by the kinase, LCK, and translocates to the cell membrane. There it remains associated with LCK in a structural complex known as the immunological synapse. Loss of PKCq in T cells produces a phenotype that is similar to loss of another important T cell protein, Notch1, suggesting these two proteins may function in the same signaling pathway. Our lab has shown that PKCq can interact with Notch1, but how this interaction regulates Notch1 function is not known. Due to the strong overlap between cellular functions regulated by PKCq and Notch1, understanding how these two proteins might function, cooperatively, can provide better insight into autoimmune diseases and may be useful in developing novel therapies We hypothesized that phosphorylation of a specific residue of PKCq (T538) is required for Notch1 cleavage and nuclear translocation. We also hypothesize that the phosphorylation status of PKCq (T538) will influence the ability of T cells to differentiate into specific T helper subsets. We used two means of inhibiting PKCq function to evaluate its regulation of Notch1 in differentiated T cells: 1) we blocked the association of PKCq and LCK, thereby preventing its movement to the immunological synapse and 2) we prevented PKCq phosphorylation on T538. We found that by preventing the phosphorylation of PKCq, we also decreased the level of cleaved Notch1 in Th1, Th2, and Th17 cells. We further determined that inhibiting PKCq decreased the amount of Notch1 that translocated to the nucleus in Th1 and iTreg cells. Understanding how PKCq interacts with and regulates Notch1 to influence T cell differentiation may lay the foundation for specifically modulating T cell responses.