SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway.
Protein kinase C-theta (PKCtheta) plays an important role in T-cell activation via stimulation of AP-1 and NF-kappaB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCtheta-interacting kinase. SPAK interacted with PKCtheta (but not with PKCalpha) 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 PKCtheta, but not by PKCalpha. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCtheta-deficient T cells. Transfected SPAK synergized with constitutively active PKCtheta to activate AP-1, but not NF-kappaB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCtheta-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 PKCtheta- and TCR/CD28-induced AP-1, but not NF-kappaB, activation. These results define SPAK as a substrate and target of PKCtheta in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-kappaB) activation.
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.
Yingqiu
Y
Li
Junru
J
Hu
Randi
R
Vita
Binggang
B
Sun
Hiroki
H
Tabata
Amnon
A
Altman
D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D018106:CD28 Antigens; D007527:Isoenzymes; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D018808:Transcription Factor AP-1; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; C076233:PRKCQ protein, human; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta