However, our previous study did not reveal the physiological pTyr-containing ligand that binds to PKC in T cells or whether this newly discovered property of the C2 domain is required for downstream effector functions of PKC, including proliferation, cytokine expression, and Th differentiation of main T cells. (NF-B) activation and Th2 differentiation, but also experienced Torin 1 a defect in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-1 (PLC1), signaling proteins that are traditionally considered to be triggered PKC-independently. These findings demonstrate that PKC takes on an important part inside a positive opinions regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important part for PKC in T cell Ca2+ signaling. This PKC-Zap70 connection could potentially serve as a encouraging and highly selective immunosuppression drug target in autoimmunity and organ transplantation. Intro T lymphocytes are a important component of the adaptive immune system. Protein kinase C- (PKC), which we have studied extensively (1), is found mainly in T lymphocytes, where it takes on nonredundant functions in cell activation, differentiation, proliferation, and survival (2, 3). PKC is unique among the PKC enzymes present in T cells because it undergoes peptide-MHC (pMHC)-induced translocation to the central region of the supramolecular activation cluster (cSMAC) region of the adult T cell immunological synapse (Is definitely) (4). There it colocalizes with the T Torin 1 cell costimulatory molecule CD28 inside a ring surrounding the central TCR (5). PKC is not globally required for all T cellCmediated reactions but, rather, has a selective part, being required Torin 1 MYO9B for Th2 (6, 7) and Th17 (8, 9) reactions, allograft rejection (10), and graft-versus-host disease (GVHD) (11, 12), but not (or considerably less) for antiviral reactions mediated by Th1 cells and cytotoxic T lymphocytes (12C15) or for graft-versus-leukemia reactions (11, 12). In contrast to the positive regulatory part of PKC in T effector (Teff) cells, PKC negatively regulates the suppressive function of regulatory T (Treg) cells (16, 17). The molecular basis for the selective and context-dependent functions of PKC in T cells is definitely unclear. Given the important and selective functions of PKC in T cells, substantial effort has been dedicated to developing selective PKC inhibitors for the treatment of autoimmune diseases, GVHD, and transplant individuals (11, 18C20). However, it has generally been hard to develop clinically useful, specific PKC catalytic inhibitors (and inhibitors of additional PKC family members in general), likely due to the similarity between PKC family members, the multiple regulatory functions that PKC enzymes play in many cells and cells, and the potential practical redundancy among PKC family members (21C23). Thus, a better understanding of the mechanisms that regulate PKC activation and downstream effector functions is needed. The intracellular localization, activation, and effector functions of PKC are regulated by complex mechanisms, including phosphorylation (24C29), sumoylation (30), protein-protein relationships (2, 3, 31), intrinsic rules by its regulatory domains (32C34), and catalytic activity (33). We have demonstrated the mechanism and importance of the localization of PKC to the cSMAC region of the Is usually by showing that this V3 (hinge) region between the regulatory and catalytic domains of PKC binds to the cytoplasmic tail of CD28, likely through the kinase Lck, and this association is critical for the downstream functions of PKC in T cells (31). We also demonstrated that, similar to its nearest relative within the PKC family, PKC (35), the N-terminal C2-like domain name of PKC is usually a phosphotyrosine (pTyr)-binding domain name (36). Nevertheless, the biological relevance of this function of PKC, as well as the identity of the pTyr-containing protein(s) that associate(s) with the PKC C2 domain name in TCR-stimulated T cells remains to be decided. To address these unresolved questions, we functionally characterized PKC-deficient T cells reconstituted with a PKC mutant that is incapable of pTyr binding and used mass spectrometry (MS) to identify a pTyr-containing ligand of the PKC C2 domain name. Here, we demonstrate that pTyr binding to the PKC C2 domain name is required for full TCR-induced, PKC-dependent downstream signaling and effector functions, identify the chainCassociated protein kinase of 70 kDa (Zap70).