Protein Kinase Cα (PKCα) Acts Upstream of PKCθ To Activate IκB Kinase and NF-κB in T Lymphocytes

Abstract

NF-B is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-B activation, an IB kinase (IKK)-dependentprocess. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKC␣, but not PKC␤1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca 2؉ influx, the catalytically active PKC␣A25E induces IKK activity and NF-B-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca 2؉ binding to PKC␣ and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKC␣ dependent and that a later phase (5 to 25 min) of IKK activation is PKC dependent. Activation of IKK-and NF-B-dependent transcription by PKC␣A25E is abrogated by the PKC inhibitor rottlerin or the expression of the kinase-inactive form of PKC. Taken together, our results suggest that PKC␣ acts upstream of PKC to activate the IKK complex and NF-B in T lymphocytes following TCR activation.Identification of the molecular events regulating T-cell activation is paramount to understanding the regulation of the immune response. The signal transduction pathways triggered by antigen presentation lead to the immediate activation of multiple transcription factors that further amplify the process of lymphocyte activation, ultimately leading to cellular proliferation and division. T-cell receptor (TCR) engagement, together with the second costimulatory signal derived from engagement of the CD28 receptor, results in NF-B activation (13,31,36). When occurring together with NF-AT, AP-1, and octomer, NF-B activation leads to interleukin-2 (IL-2) expression (17,27,30).NF-B is a heterodimer of transcription factors that belong to the Rel family of proteins. The canonical NF-B is a heterodimer of p65 (RelA) with p50 or p52 (35,50,54). This heterodimer is anchored by a group of proteins named IB, which function to retain NF-B in the cytosol by masking its nuclear localization signal (1,4,45,60). IB␣ is the prototype IB molecule known to control the subcellular localization of NF-B (p50/p65). Following activation of certain signal transduction pathways, a site-specific hyperphosphorylation of IB␣ at S32 and S36 renders the inhibitor molecule susceptible to site-specific ubiquitination and subsequent degradation by the proteasome complex (8,9,18,62,68). This releases NF-B, allowing it to undergo nuclear translocation. Two IB␣ kinases, IKK␣ (19,52,70) and IKK␤ (46), which are contained within a high-molecular-weight complex, target the phosphorylation of S32 and S36 of IB␣ following stimulation by various stimuli. While...

Activation of Nuclear Factor κB by Somatostatin Type 2 Receptor in Pancreatic Acinar AR42J Cells Involves Gα14 and Multiple Signaling Components

“…In addition, different isoforms of PKC have been shown to participate in NFB signaling. Modulation of IKK activity by the Ca 2ϩ -sensitive PKC␣ is clearly evident in T lymphocytes (27), whereas macrophages from PKC⑀-deficient mice display an impaired IKK response upon stimulation by lipopolysaccharide (28). Moreover, physical association with the IKK complex has been demonstrated for PKC (29).…”

A mosaic de novo duplication of 17q21–25 is associated with GH insensitivity, disturbed in vitro CD28-mediated signaling, and decreased STAT5B, PI3K, and NF-κB activation

“…Interestingly, overexpression of PKCd has been associated with decreased tyrosine phosphorylation and increased serine phosphorylation at the insulin receptor, and it causes internalization of the insulin receptor (37). Furthermore, PKCa has been implicated in the activation of NF-kB downstream of TCR/CD28-induced T-cell activation (38), and it can also play an inhibitory role during the regulation of the cell cycle (33). With respect to PI3K signaling, PKCa, even at physiological concentrations, serves as an endogenous negative feedback inhibitor of insulin signaling through IRS1, PI3K, PKB, and PKCl to the glucose transport system in both skeletal muscles and adipocytes (39), which is in line with the decreased PI3K phosphorylation in response to GH in the patient's fibroblasts.…”

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy

“…The two members of the atypical PKC subfamily (PKCz and PKCl/i) are involved in the control of NF-kB through IKKb activation Sanz et al, 1999). The conventional PKC isoform PKCa, but not PKCb1, is also required for the activation of the IKK complex upon T-cell activation triggered by CD3/CD28 crosslinking (Trushin et al, 2003). Another recent study has proposed an elegant model in which TNF-a binding to the TNFR1 activates PI-PLCg to induce PKCa and c-Src activation leading to tyrosine phosphorylation of IKKa/b (Huang et al, 2003).…”