SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

Abstract

Protein kinase C-h (PKCh) plays an important role in T-cell activation via stimulation of AP-1 and NF-jB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCh-interacting kinase. SPAK interacted with PKCh (but not with PKCa) 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 PKCh, but not by PKCa. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCh-deficient T cells. Transfected SPAK synergized with constitutively active PKCh to activate AP-1, but not NF-jB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKChinteracting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAKspecific RNAi or a dominant-negative SPAK mutant inhibited PKCh-and TCR/CD28-induced AP-1, but not NF-jB, activation. These results define SPAK as a substrate and target of PKCh in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-jB) activation.

MicroRNA-34 mediates AR-dependent p53-induced apoptosis in prostate cancer

“…The expression of endogenous SPAK was inhibited by infection with recombinant lentivirus constructs pLSL-puro expressing siRNA hairpin under control of the RNA H1 promoter. 3 The structures of two SPAK gene-specific oligonucleotides complementary to human SPAK mRNA 21 were as follows: 5'-CCCAGGCAAGAACGTGTA-3' and 5'-ATTCAAGC-CATGAGTCAGT-3' corresponding to nucleotides 255-273 and 334-352 respectively. LNCaP was infected with lentiviruses either with one si-SPAK or with mixture of two (labeled DB).…”

Germinal center kinases in immune regulation

“…During T-cell activation, SPAK is involved in TCR/CD28-induced AP-1 activation through interactions with the PKCh molecule via its C-terminal region. 108 Receptor expressed in lymphoid tissues and its homologs RELL1 and RELL2 are non-canonical members of TNFR family that may be distinct from those of classical members in terms of signaling. The C-terminal domain of SPAK/OSR1 may bind to the 'RFRV' motif found in the cytoplasmic domain of receptor expressed in lymphoid tissue subfamily members, leading to phosphorylation of receptor expressed in lymphoid tissue and downstream signaling.…”

Crystal structure of domain‐swapped STE20 OSR1 kinase domain

“…4,7 OSR1 and SPAK have been suggested to be coupled to cellular events such as cell differentiation, cytoskeleton rearrangement, cell proliferation, and transformation. 3,[11][12][13][14][15] In addition, extensive biochemical and physiological studies demonstrate that OSR1 and SPAK are also involved in the regulation of ion homeostasis and volume control in mammalian cells. For example, during hyperosmotic stress, OSR1 and SPAK interact with and activate NKCC1 (Na…”