Regulation of Protein Kinase Cθ Function during T Cell Activation by Lck-mediated Tyrosine Phosphorylation

Abstract

Protein kinase C (PKC) is a novel Ca2؉ -independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR)⅐CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKC is tyrosine phosphorylated in Jurkat T cells upon TCR⅐CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKC. Lck phosphorylated and was associated with the regulatory domain of PKC both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Srchomology 2 and Src-homology 3 domains of Lck contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKC was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKC (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKC, which may in turn modulate the physiological functions of PKC during TCR-induced T cell activation. Protein kinase C (PKC)1 is a family of serine/threonine kinases that play critical roles in the regulation of differentiation and proliferation in many cell types and in the response to diverse stimuli (reviewed in Refs. 1 and 2). Products of the 10 known mammalian PKC genes are classified into four subfamilies of Ca 2ϩ -dependent (or conventional, PKC␣, -␤, and -␥), Ca 2ϩ -independent (or novel, PKC␦, -⑀, -, and -), atypical (PKC and /), and PKC/D enzymes. Activity of PKC enzymes is regulated by phosphorylation and binding of defined cofactors. Enzyme activation is associated with its redistribution among different cellular compartments, commonly from the cytosolic to the particulate (membrane) fraction. Studies indicate that PKC is also important during T cell activation. This is indicated by the ability of physiological T cell receptor (TCR) ligands to activate PKC and induce its translocation from the cytosol to the particulate fraction; by the ability of PKC inhibitors, or PKC depletion by prolonged phorbol ester treatment, to block lymphocyte signaling and activation; by the requirement for persistent PKC activation during mitogenic T cell activation; and, finally, by the diminished TCR⅐CD3-mediated proliferation in PKC-deficient T cells (reviewed in Ref. 3).PKC is a novel Ca 2ϩ

Cutting Edge: CD28-Mediated Transcriptional and Posttranscriptional Regulation of IL-2 Expression Are Controlled through Different Signaling Pathways

“…Under these conditions, downstream signaling from PKC , as detected by NF-B activation and upregulation of IL-2 transcription, is attenuated. This correlation between PKC localization to the c-SMAC and PKC function suggests that the major function of CD28 in this pathway may be to recruit PKC to the c-SMAC, allowing for PKC interaction with upstream regulators, such as Lck (20), or downstream activators of NF-B, such as the Bcl10/Malt1/Carma complex (21,22).…”

PKCθ mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia

“…These observations are consistent with the previously shown ability of pre-TCR to colocalize with the p56 Lck Src kinase into glycolipid-enriched membrane domains (rafts), apparently without any need for ligand (SaintRuf et al, 2000). On the other hand, p56 lck , known to induce tyrosine phosphorylation of PKCy, may in turn modulate the physiological functions of PKCy during TCR-induced T-cell activation (Liu et al, 2000), suggesting that pre-TCR membrane localization and PKCy activation are regulated by the same kinase (i.e. Lck).…”

“…PKCy translocation to the cell membrane is increased in Notch3-IC transgenic thymocytes and depends on the presence of pTa/pre-TCR It has been previously shown that ligand-dependent triggering of TCR is associated with membrane translocation of PKCy, which colocalizes with the TCR/CD3 complex and requires phosphorylation by and physical association with p56 Lck Src kinase (Monks et al, 1997;Liu et al, 2000). In contrast, pre-TCR has been shown to translocate and colocalize with the p56 Lck into glycolipid-enriched membrane domains (rafts), apparently without any need for ligation (Saint-Ruf et al, 2000).…”

Protein kinase C-θ regulates KIT expression and proliferation in gastrointestinal stromal tumors

“…PKCy:NFkB interactions can be bidirectional, as in the case of T-cell receptor (TCR)-induced NFkB activation, regulated by 3-phosphoinositidedependent kinase 1, which can activate PKCy, resulting in PKCy recruitment to lipid rafts (Lee et al, 2005). TCR pathways also regulate PKCy tyrosine phosphorylation, which is mediated by SRC family protein tyrosine kinases such as LCK (Liu et al, 2000). In addition, PKCy can regulate gene expression, functioning as a positive modulator of retinoid X receptorresponsive element (RXRE)-dependent transcription during T-cell activation (Ishaq et al, 2002).…”