No Specific Subcellular Localization of Protein Kinase C Is Required for Cytotoxic T Cell Granule Exocytosis

Abstract

Cytotoxic T cells kill virus-infected cells and tumor cells by releasing lytic granules that contain cell-killing contents. Exocytosis requires calcium influx and protein kinase C (PKC) activation. Here, we extend our previous finding regarding the lack of isoform specificity of PKCs in the granule release step, showing that mutant constitutively active PKC␦ can substitute for phorbol esters and support exocytosis. PKC␦, a novel PKC isoform, was recently shown to play a role in lytic granule reorientation. Surprisingly, however, our results suggested that mutant PKC␦ did not localize to the plasma membrane (PM). To test directly whether PKC has to be in the PM to drive exocytosis, we generated mutants of various PKC isoforms that were tethered either to the outer mitochondrial membrane or to the PM. Tethered mutant PKC␦s were able to promote exocytosis as effectively as the untethered version. The substrates of PKCs involved in lytic granule exocytosis are currently unknown, but subcellular localization is believed to be a critical factor in determining PKC accessibility to substrates. That there is no requirement for specific PKC localization in lytic granule exocytosis may have important implications for the identity of PKC substrates.

Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells

“…Thus, PKCδ kinase activity appears to be necessary for the positive effect of PKCδ on MVB polarization. In addition, we observed that GFP-PKCδ underwent activation upon IS formation, as assessed by WB analysis of T505 phosphorylation (3–4 fold induction) at the activation loop of GFP-PKCδ (Figure 2E) (34), in agreement with PKCδ activation described in mouse CTL (28). Altogether, these data indicate that PKCδ is required for the polarization of both MVB and the MTOC toward the IS in T lymphocytes.…”

“…Plasmids used in this study were as follows: pEFbos-GFP was described previously (13, 23); pEFGFP-C1bosCD63 and pECFP-C1CD63 were provided by G. Griffiths; mouse pEGFP-PKCδwt (GFP-PKCδWT), pEGFP-PKCδDR144/145A constitutively active mutant (GFP-PKCδCA) (32) and pEGFP-PKCδK376A kinase-dead mutant (GFP-PKCδKD) (33, 34) were obtained from A. Zweifach and D. M. Reyland. GFP-C1bPKCθ expression plasmid was kindly provided by I. Mérida; UpwardDAG2 (U.DAG2) (35) was generously provided by A.M. Quinn (Montana Molecular Inc.).…”

NKG2D Costimulates Human Vγ9Vδ2 T Cell Antitumor Cytotoxicity through Protein Kinase Cθ-Dependent Modulation of Early TCR-Induced Calcium and Transduction Signals

“…A recent study identified PKCd as an active constituent of the TCR signaling pathway that selectively induces movement of lytic granules in mouse CTLs (43). However, no specific subcellular localization of PKC seems to be required for cytolysis-associated exocytosis (44). The fact that NKG2D triggering accelerates Ca 2+ responses through PKCu activation and that its inhibition alters Vg9Vd2-Ca 2+ responses, therefore supports an implication of PKCu in the control of Ca 2+ responses within activated gd T cells.…”

Flow Cytometry Enables a High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis

“…As the activation of PKC is known to participate in activating ERK when cells are stimulated with PMA 12 , we tested whether 9f inhibits PKC activity. We stained fixed and permeabilized cells with an antibody that detects proteins that are phosphorylated on the consensus PKC substrate site, an approach we have used before 25 . The stimulation-dependent increase in staining with this antibody in cells treated with compound 9f was not significantly different than in DMSO-treated control cells (data not shown), suggesting that the effects of 9f lie downstream of PKC activation.…”