Protein kinase C-0 (PKC0) 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 PKC0-interacting kinase. SPAK interacted with PKC0 (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 PKC0, but not by PKC0. 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 PKC0 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 PKC0-and TCR/CD28-induced AP-1, but not NF-jB, activation. These results define SPAK as a substrate and target of PKC0 in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-jB) activation.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) or of Akt induces myoblast differentiation. Activation of p38 MAP kinase also triggers myogenic differentiation. The current paper shows that PI 3-kinase and p38 MAP kinase signalling are activated by two separate pathways during myogenic differentiation; both are required for muscle differentiation. p38-induced myogenic differentiation can be inhibited by the PI 3-kinase inhibitor LY294002 without affecting p38 activity. Similarly, a constitutively active form of Akt, myristylated c-Akt (Myr-Akt), induces myogenic differentiation that is inhibited by the p38 inhibitor SB203580. An analysis of the two forms of p38, p38 and p38β, shows that the activity of both is required for myogenic differentiation. These data suggest that PI 3-kinase and p38 signalling are essential and parallel pathways for myogenic differentiation. They may either affect different downstream targets required for myogenesis or they may converge on shared targets that require input from both signalling pathways.

A novel protein kinase whose activity can be stimulated by mitogen in vivo was cloned and characterized. The cDNA of this gene encodes an 802-amino acid protein (termed RLPK) with the highest homology (37% identity) to the two protein kinase families, p90RSK and p70RSK. Like p90RSR, but not p70RSK, RLPK also contains two complete nonidentical protein kinase domains. RLPK mRNA is widely expressed in all human tissues examined and is enriched in the brain, heart, and placenta. In HeLa cells, transiently expressed epitopetagged RLPK can be strongly induced by epidermal growth factor, serum, and phorbol 12-myristate 13-acetate, but only moderately up-regulated by tumor necrosis factor-a and other stress-related stimuli. The activity of RLPK stimulated by epidermal growth factor was not inhibited by several known protein kinase C inhibitors nor by rapamycin, a known specific inhibitor for p70RSK, but could be inhibited by herbimycin A, a tyrosine kinase inhibitor, and partially inhibited by PD98059 or SB203580, inhibitors for the mitogen-activated protein kinase pathways. Recombinant RLPK possesses high phosphorylation activity toward histone 2B and the S6 peptide, RRRLSSLRA. Although purified recombinant RLPK can be phosphorylated by ERK2 and p38a in vitro, its activity is not affected by this phosphorylation. Moreover, the treatment of RLPK with acid phosphatase did not reduce its in vitro kinase activity. These data suggest that RLPK is structurally similar to previously isolated RSKs, but its regulatory mechanism may be distinct from either p70RSK or p90RSKs.