TAO (Thousand-and-one Amino Acid) Protein Kinases Mediate Signaling from Carbachol to p38 Mitogen-activated Protein Kinase and Ternary Complex Factors

Abstract

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein G␣ o could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant G␣ o Q205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser 383 , and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus. TAO11 (thousand-and-one amino acids 1) and TAO2 are protein kinases that were originally identified based on their similarity to the yeast p21-activated protein kinase Ste20p, a protein kinase upstream in a mitogen-activated protein kinase (MAPK) pathway in yeast (1, 2). JIK is a third TAO-like kinase (3). The Ste20p family contains a diverse array of protein kinases, several of which have been shown to act upstream of MAPKs (4). TAO1 and TAO2 each contain over 1000 residues with catalytic domains at their N termini. TAOs activate MAPK pathways because they have MAP kinase kinase kinase (MAP3K) activity; they phosphorylate the p38-activating kinases called MAP/ERK kinases (MEKs, also known as MKKs or MAP2Ks) 3 and 6, which then phosphorylate p38 (1, 2, 5). The preferential phosphorylation of these two MEKs arises in part because TAOs dock to these MEKs through a region Cterminal to the TAO kinase domain (2, 5).Ternary complex factors (TCFs) are among the transcription factors under the control of MAPKs (6 -12). Upon phosphorylation by MAPKs, TCFs form a complex with the serum response factor at the serum response element, thereby stimulating the transcription of c-fos and other genes containing this element. The TCFs, Elk1, Sap1, and Sap2, contain a conserved C-terminal transactivation domain with multiple (S/T)P motifs, which are minimal consensus sites for MAPK phosphorylation. Elk1 can be phosphorylated by at least three MAPK subgroups, ERK1/2, c-Jun-N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), and p38. In contrast, Sap1 can be phosphorylated by ERK1/2, ERK5, and p38, but not JNK/ SAPKs (7, 12, 13). We explored downstream actions of TAO2 by examining its effects on transactivation of TCFs. Cotransfection studies and reporter assays demonstrated that TAO2 enhances phosphorylation and transactivation of TCFs in 293 cells. TAO2-dependent transactivation of TCFs was inhibited by the p38 selective inhibitor SB203580, suggest...

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