Abstract

Protein kinase C- (PKC) plays an important role in T-cell activation via stimulation of AP-1 and NF-B. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKC-interacting kinase. SPAK interacted with PKC (but not with PKC) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKC, but not by PKC. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKC-deficient T cells. Transfected SPAK synergized with constitutively active PKC to activate AP-1, but not NF-B. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKC-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKC- and TCR/CD28-induced AP-1, but not NF-B, activation. These results define SPAK as a substrate and target of PKC in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-B) activation.