The Physical Association of Protein Kinase Cθ with a Lipid Raft-Associated Inhibitor of κB Factor Kinase (IKK) Complex Plays a Role in the Activation of the NF-κB Cascade by TCR and CD28

摘要

We investigated the role of protein kinase C θ (PKCθ) in the activation of the NF-κB cascade in primary human CD4+ lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCθ participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IκB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca2+-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCθ physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4+ T cells. The same set of stimuli also induced relocation of endogenous PKCθ and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IκBα sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCθ and IKKβ colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCθ activated IKKβ in Jurkat T cells. Expression of dominant-active PKCθ also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCθ by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.