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

Zhu Chen {ddagger} §, Malavika Raman {ddagger}, Linda Chen , Sheu Fen Lee, Alfred G. Gilman and Melanie H. Cobb ||

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, February 3, 2003 , and in revised form, March 24, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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{alpha}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{alpha}oQ205L. 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, Ser383, 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 C-terminal to the TAO kinase domain (2, 5).

Ternary complex factors (TCFs) are among the transcription factors under the control of MAPKs (612). 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, suggesting that p38 is the key mediator of this action.

MAPKs have readily been linked to ligand-dependent biological processes through the use of pharmacological inhibitors and gene-disruption experiments (14). Some of the earliest studies of p38 demonstrated that it was required for the translation of mRNAs encoding certain inflammatory cytokines in response to lipopolysaccharide, for example (15). Biochemical and overexpression experiments have implicated at least six different MAP3Ks in the regulation of p38; however, it is unclear which or how many of these kinases mediate the action of any ligand (1, 2, 1624). The activity of endogenous TAO, like that of many MAP3Ks, has been difficult to measure. Therefore, overexpression and in vitro analysis have been the primary means used to probe the functions of the TAO protein kinases. Because our studies have shown that TAOs stimulate p38, we searched previously for TAO2 regulators among stress stimuli linked to p38 activity and found that TAO2 activity was increased from 1.5- to 3-fold by nocodazole, sodium chloride, and sorbitol (5). Here we report that the muscarinic agonist carbachol modestly stimulates TAO2 activity and also enhances p38 activity in 293 cells. To determine whether TAOs mediate activation of p38 by carbachol, we used dominant interfering kinase mutants and found that MEKs 3 and 6 and TAOs were required for p38 activation not only by carbachol but also by a constitutively active mutant of G{alpha}o. These findings suggest that TAOs are the intermediates that link certain heterotrimeric G protein-coupled receptors (GPCRs) to the p38 MAPK pathway.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Reagents—Expression plasmids for Gal-Elk (the Gal4 DNA-binding domain fused to the Elk1 transactivation domain) and Gal-lu (Gal4-driven luciferase) were as described (25). Constitutively active mutants of MEK6 (MEK6DD), and Raf-1 (Raf BXB), and the thymidine kinase-driven renilla luciferase (pRL-TK) were generously provided by Jennifer Swantek. pCMV5-(His)6-Elk1 was as described (26). pSG-Gal-Sap (the Gal4 DNA-binding domain fused to the Sap1 transactivation domain) was kindly provided by Peter Shaw (Nottingham, UK). G{alpha}o,G{alpha}oQ205L, G{alpha}i1,G{alpha}i1 Q204L, G{alpha}q, and G{alpha}qQ209L were as described (27, 28). TAO1 constructs were as described (1). A D169A mutation was created in pCMV5-HA-TAO1 (1–416) with the QuikChange kit from Stratagene. TAO2 constructs were as described (2, 5). TAO2-(1–993) was used because full-length TAO2 was expressed very poorly. pCMV5-M2 (M2 muscarinic receptor) was kindly provided by Elliot Ross. Site-directed mutagenesis was performed on pCMV5-Myc-MEK3, pSR{alpha}-HA-MEK6, and pCS3+-MT-Myc-MEK7 plasmids to generate the mutations of the ATP binding Lys to Met, thereby creating constructs expressing kinase-deficient enzymes (5). Expression plasmids for MEK1KM and MEK5KM were as described (29, 30). The p38 inhibitor SB203580 was purchased from Calbiochem and prepared as a 20 mM solution in dimethyl sulfoxide (Me2SO). Antibodies that recognize Elk1, phospho-Elk1, and phospho-ERK1/2 were obtained from New England Biolabs and used for immunoblotting under the recommended conditions. Polyclonal antisera C20 (from Santa Cruz Biotechnology) and P287 (31) were used to immunoblot and immunoprecipitate p38, respectively. Anti-TAO2 antibodies were as described (5).

Cell Treatment, Transfections, Reporter Assays, and Kinase Assays— Endogenous TAO2 was immunoprecipitated from lysates of 293 cells that had been serum-deprived for 18 h and then left untreated or exposed to 60 ng/ml insulin, 10 µM carbachol, 10 µM epinephrine, 10 µM isoproterenol, or 0.4 M sorbitol for 5 or 10 min. Some cells were treated with either 100 nM or 10 µM nicotine for either 1 or 10 min. The effects of 500 nM bradykinin at 5 and 15 min were also examined. TAO2 activity was assayed with MEK6KM as in Ref. 5. To examine the activities of endogenous p38, 3 µg of one or more of the following constructs encoding the G proteins listed above, the TAOs, and the positive and negative controls, were transfected as indicated into 293 cells cultured on 60 mm dishes at about 80% confluence using calcium phosphate as described (5). Alternatively, 3 µg of plasmids encoding G{alpha}oQ205L or pCMV5-M2 were cotransfected with 4 µg of a panel of kinase-dead constructs or empty vector control. 16 h after transfection, cells were serum-starved for another 24 h and then harvested in detergent lysis buffer (5). Transfection efficiency was usually >40% under these conditions. Carbachol (10 µM) was added for 10 min prior to harvest. Endogenous p38 was immunoprecipitated from ~0.2 mg total lysate protein and assayed using 0.3 µg of GST-ATF2 as the substrate (5). To study the phosphorylation of Elk1 in cells, 1 µg of pCMV5-(His)6-Elk1 was transfected into 293 cells with 3 µg of vector control or TAO2 constructs as described earlier. To examine the transactivation of Elk1 or Sap1, 293 cells were grown on 35-mm wells to ~70% confluence and then transfected with 0.5 µg of Gal-Elk or Gal-Sap, and 0.5 µg of Gal-lu, 0.25 µg of pRL-TK (as an internal control for transfection efficiency) plus 1.5 µg of empty vector or vector encoding Raf BXB, MEK6DD, or TAO2. 24 h after transfection, cells were treated with either Me2SO or 20 µM SB203580 and serum-starved for another 24 h before harvesting. Cell lysates were subjected to dual luciferase assays performed according to the manufacturer's protocol using a luciferase assay kit and a Turner luminometer (Promega). Relative luciferase activity was determined as the ratio of Gal4-driven firefly luciferase activity to TK-driven renilla luciferase activity. Data were plotted as fold activation compared with that induced by the empty vector control from triplicate measurements.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Activities of Endogenous TAO2 and p38 Are Stimulated by Carbachol—We screened a series of ligands in an attempt to identify a pathway in which TAO2 might be the MAP3K that regulates p38. C2C12 cells were untreated or treated with carbachol, sorbitol, insulin, epinephrine, or isoproterenol. TAO2 was immunoprecipitated from cell lysates and assayed with kinase-dead MEK6 as substrate. Of the ligands tested here, only carbachol and sorbitol, a previously identified activator (5), increased TAO2 activity >1.5-fold (Fig. 1A); activity was highest after 5 min and decreased by 10 min of treatment with either carbachol or sorbitol. Carbachol also stimulated TAO2 activity in 293 and HeLa cells, whereas other ligands, including bradykinin, did not (data not shown). TAO2 autophosphorylation and a decrease in its electrophoretic mobility corresponded well with activity at 5 min. Nicotine did not activate TAO2, although ERK1/2 were activated under these conditions (Fig. 1B). The ability of carbachol but not nicotine to activate TAO2 suggests that this effect is mediated by a muscarinic receptor.



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FIG. 1.
Effects of ligands on the activity of endogenous TAO2. A, C2C12 cells were untreated or treated with 10 µM carbachol, 10 µM isoproterenol, 60 ng/ml insulin, 10 µM epinephrine, or 0.4 M sorbitol for 5 or 10 min. Cells were lysed, and TAO2 was immunoprecipitated and assayed with kinase-dead MEK6 as substrate. Bottom panel, autoradiogram of MEK6 phosphorylation by TAO2. Top panel, autoradiogram of TAO2 autophosphorylation. One of five similar experiments. B, C2C12 cells were untreated with 100 nM or 10 µM nicotine for 1 or 10 min. Top panel, TAO2 autophosphorylation is shown graphically. Middle panel, autoradiogram of MEK6 phosphorylation by TAO2. Bottom panel, immunoblot of lysates with antibodies to phosphorylated, active ERK1/2. One of four similar experiments.

 

In C2C12 or 293 cells, treatment for 10 min with carbachol also weakly stimulated p38 activity 1.5–2-fold (data not shown and Fig. 2A, lanes 1 and 2). When the M2 muscarinic receptor was overexpressed in 293 cells, carbachol caused a significantly greater activation of p38 than in cells not over-expressing the receptor (Fig. 2A, lane 4).



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FIG. 2.
Effect of inhibitory kinase mutants on activation of endogenous p38 by carbachol. A, 293 cells were transfected with either empty vector or pCMV5-M2. Cells were untreated or stimulated with carbachol for 10 min prior to harvest. Activity of endogenous p38 was assayed with ATF2 as substrate. Top panel, autoradiogram showing ATF2 phosphorylation by p38. Bottom panel, anti-p38 immunoblot as protein expression control. B, 293 cells were transfected with constructs encoding the indicated proteins. Cells were unstimulated or treated with carbachol for 10 min prior to harvest. Kinase activities (top panel) and expression levels (bottom panel) of endogenous p38 as for panel A. One of three representative experiments is shown.

 

TAO, MEK3, and MEK6 Are Required for Activation of Endogenous p38 by Carbachol—To determine whether activation of p38 by carbachol is mediated by TAOs, the effects of the expression of kinase-dead TAO and MEK mutants were examined (Fig. 2B). Coexpression of kinase-dead forms of MEK3, MEK6, or full-length TAO1 reduced activation of p38 by carbachol to less than half that in cells not expressing these mutant kinases (Fig. 2B, lanes 6, 8, and 10). Truncated, kinase-dead mutants of TAO1 and TAO2 failed to block stimulation by carbachol (Fig. 2B, lanes 12 and 14), indicating a requirement of the C-terminal domain of TAO for a blockade of p38 activation. In several experiments, expression of these truncated inactive kinases slightly enhanced both basal and carbachol-dependent p38 activity; this observation is explored further below. These results indicate that TAOs are required for activation of p38 by carbachol.

Active G{alpha}o Activates Endogenous p38 in 293 Cells—We tested the effect of G{alpha}i1, G{alpha}q and G{alpha}o on the activity of endogenous p38 by transfecting cells with cDNAs encoding constitutively active forms of the G proteins, TAOs, or other potential p38 activators. Cell lysates were immunoblotted with the p38 antibody to demonstrate that its expression was unchanged (Fig. 3, bottom panel). Neither G{alpha}q nor G{alpha}i1 stimulated the activity of p38 and were not examined further (data not shown). Full-length TAO1 (Fig. 3, lane 3), and truncated forms of TAOs, TAO1-(1–416) (Fig. 3, lane 4) and TAO2-(1–451) (Fig. 3, lane 8), enhanced p38 activity ~3-fold; neither kinase-dead TAO1 (Fig. 3, lane 2) nor kinase-dead TAO2 (Fig. 3, lane 7) alone affected p38 activity. Neither Raf BXB (Fig. 3, lane 10) nor wild-type G{alpha}o (Fig. 3, lane 6) had any effect on p38 activity, but the constitutively active G{alpha}o mutant activated p38 by ~3-fold (Fig. 3, lane 9). Thus, active G{alpha}o activates endogenous p38 in 293 cells.



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FIG. 3.
G{alpha}o Q205L and TAO activate endogenous p38 in 293 cells. 293 cells were transfected with the indicated constructs. After 16 h, serum was removed for 24 h prior to cell harvest. Endogenous p38 was immunoprecipitated from the lysates and assayed as described in the Fig. 2 legend. Top panel, autoradiogram showing phosphorylation of ATF2. Bottom panel, anti-p38 immunoblot of cell lysates. One of four representative experiments is shown.

 

Full-length Kinase-dead TAO1 Blocks Activation of Endogenous p38 by Active G{alpha}oWe wished to determine whether G{alpha}o and TAO function in the same pathway to activate p38. To address this question, we tested the effects of kinase-dead mutants of the potential kinase mediators of G{alpha}o on activation of p38. TAO2-(1–451) (Fig. 4A, lane 3) and G{alpha}oQ205L alone (Fig. 4, lane 6) each enhanced the activity of endogenous p38 by ~3–4-fold, as shown above. When G{alpha}oQ205L was cotransfected with kinase-defective forms of full-length TAO1 (Fig. 4, lane 7), MEK3 (Fig. 4, lane 9), or MEK6 (Fig. 4, lane 11), the activation of p38 was reduced to no more than 1.2-fold. In contrast, kinase-dead forms of MEKs 1, 5, 7, and truncated TAO2 failed to interfere with the activation of p38 by G{alpha}oQ205L (Fig. 4, lanes 8, 10, 12, and 13). Kinase-dead MEKK1, either full-length or the kinase domain alone, did not block p38 activation by the G protein (data not shown). These results suggest that G{alpha}oQ205L activates p38 through TAOs and MEK3/6.



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FIG. 4.
Kinase-defective forms of TAO, MEK3, and MEK6 block activation of endogenous p38 by G{alpha}oQ205L. A, 293 cells were transfected with empty vector control or MEKK as a positive control, TAO2 constructs, or G{alpha}oQ205L in combination with the indicated constructs encoding kinase-defective enzymes. Endogenous p38 was immunoprecipitated from the cells and assayed as described in the Fig. 2 legend. Immunoblotting indicated that equal amounts of p38 were present in the cell lysates (bottom panel). B, 293 cells were transfected with empty vector control or G{alpha}oQ205L in combination with kinase-dead TAO constructs. Top panel, activity of endogenous p38 was assayed as in for panel A. Bottom panel, immunoblot of cell lysates indicating that comparable amounts of p38 were present. One of three representative experiments is shown.

 

The C-terminal Regulatory Domain of TAO Is Required for the Blockade of p38 Activation—We noted that the defective form of full-length TAO1 blocked p38 activation by G{alpha}oQ205L, whereas the truncated TAO2 mutant failed to do so. To determine whether the blockade required the C-terminal domains of TAO proteins, the capacities of kinase-defective forms of full-length and truncated (1–416) TAO1 (full-length TAO2 could not be expressed under these conditions) to inhibit activation of p38 by G{alpha}oQ205L were compared (Fig. 4B). Unlike the full-length form of TAO1, truncated kinase-dead TAO1 lost the ability to block stimulation of p38 activity by G{alpha}oQ205L. Thus, the C-terminal regulatory domain of TAOs is apparently required for inhibition. One possible explanation is that the C termini of TAO1 and TAO2 harbor a domain that interacts with an additional protein required for a TAO activation complex; the proposed interaction may be required for efficient blockade.

TAO2 Stimulates Phosphorylation of Elk1 on the Major Phosphoacceptor Site—To determine whether TAO2 enhances the phosphorylation of the TCF Elk1, pCMV5-(His)6-Elk1 was transfected into 293 cells with either the empty vector control or various TAO2 constructs. Cell lysates were blotted with either an anti-Elk1 antibody to recognize total Elk1 protein (Fig. 5, bottom panel) or an antibody that specifically recognizes phosphorylated Ser383, the major site of activating phosphorylation of Elk1 (Fig. 5, top panel) (1, 2). TAO2-(1–993), TAO2-(1–320), and TAO2-(1–451) all stimulated Elk1 phosphorylation. Catalytically defective TAO2-(1–993) failed to stimulate phosphorylation, indicating that TAO2 kinase activity is required.



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FIG. 5.
TAO2 enhances phosphorylation of Elk1 on Ser383. pCMV5-(His)6-Elk1 was transfected into 293 cells with either pCMV5-Myc as a control or the indicated TAO2 constructs. 30 µg of lysate proteins were subjected to SDS-PAGE and immunoblotting using antibodies that recognize either total Elk1 (bottom panel) or Elk1 phosphorylated on Ser383 (top panel). One of four representative experiments is shown.

 

TAO2 Stimulates Transactivation by Elk1 and Sap1 Fusion Proteins in Part through p38 —The enhanced in vivo phosphorylation of Elk1 by TAO2 indicated that TAO2 may stimulate transactivation of TCFs through MAP kinases. To test this hypothesis, a chimeric construct composed of the Gal4 DNA-binding domain and the Elk1 C-terminal transactivation domain (Gal-Elk) was cotransfected with a Gal4-driven luciferase construct (Gal-lu) and TAO constructs into 293 cells (Fig. 6A). Raf BXB, a constitutive activator of the ERK1/2 pathway, stimulated transactivation of Elk1 to a great extent, presumably through ERK1/2. TAO2-(1–320) and TAO2-(1–451) also stimulated Elk1 transactivation.



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FIG. 6.
TAO2-stimulated transactivation of Elk1 and Sap1. A, 293 cells were transfected with Gal-Elk, Gal-lu, and either empty vector or plasmids encoding Raf BXB or the indicated forms of TAO2. pRL-TK was also transfected as an internal control for transfection efficiency. The result of one of three similar reporter assays, matched to data in panel B, is shown. B, 40 µg of lysate proteins from panel A were subjected to SDS-PAGE and immunoblotted to detect either total ERK1/2 (bottom panel) or phosphorylated, active ERK1/2 (top panel). C, 293 cells were transfected with Gal-Sap1, Gal-lu, and either empty vector or plasmids encoding Raf BXB or TAO2-(1–320). The result of one of three similar reporter assays, matched to data in panel D, is shown. D, 40 µg of lysate proteins from panel C were immunoblotted to detect either total ERK1/2 proteins (bottom panel) or phosphorylated, active ERK1/2 (top panel).

 

We showed previously that TAO2 activates endogenous p38, but not JNK/SAPK or ERK1/2, in 293 cells (5), suggesting that TAO signals to TCFs through p38. To confirm that ERK1/2 are not involved in the effect of TAO2 on Elk1 transactivation, lysates from cells that had been subjected to reporter assays (Fig. 6A) were immunoblotted with antibodies to detect total ERK1/2 proteins and active ERK1/2 proteins (Fig. 6B). Raf BXB greatly enhanced the amount of active ERK1/2 relative to the control. In contrast, in TAO2-transfected cells, even though transactivation of Elk1 was stimulated to a comparable extent as by Raf BXB, no active ERK1/2 were detected. PD98059, which blocks activation of ERK1/2, did not inhibit TAO-mediated Elk1 transactivation (data not shown).

We also examined the effects of TAO2 on transactivation of the TCF, Sap1. Sap1 can be activated by ERK1/2 and p38, but not JNK/SAPKs. A Gal4-Sap1 fusion construct (Gal-Sap) was cotransfected with Gal-lu and either Raf BXB or TAO2-(1–320) into 293 cells (Fig. 6C). As expected, Raf BXB greatly stimulated transactivation of Sap1. TAO2-(1–320) also stimulated Sap1 transactivation. To confirm that ERK1/2 are not involved in the effect of TAO2 on Sap1 transactivation, lysates tested for reporter activity (Fig. 6C) were immunoblotted with antibodies to detect active ERK1/2 proteins (Fig. 6D). Again, active ERK1/2 were detected in lysates from cells expressing Raf BXB, but not in those expressing TAO2, indicating that ERKs are not involved in the effect of TAO2 on Sap1.

We used the p38 inhibitor SB203580 (15) to determine whether p38 is required for the activation of TCFs by TAO2 (Fig. 7, A and B). As expected, stimulation of Elk1 and Sap1 transactivation by Raf BXB was unaffected by SB203580. MEK6DD, a constitutively active mutant of MEK6, which is upstream of p38, enhanced Elk1 and Sap1 transactivation ~5–7-fold. In both cases, activation by MEK6DD was partially reduced by SB203580. The effects of TAO2-(1–320) and TAO2-(1–451) on Elk1 and Sap1 transactivation were also partially inhibited by SB203580, suggesting that p38 mediates, at least in part, TCF transactivation. Consistent with the expectation that its kinase activity is required for TCF activation, TAO2-(1–451)DA failed to stimulate transactivation of Elk1 or SAP1.



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FIG. 7.
TAO2-stimulated transactivation of Sap1 is mediated by p38. A, 293 cells were transfected with Gal-Elk1, Gal-lu, and either empty vector or plasmid encoding Raf BXB, MEK6DD, or the indicated forms of TAO2. pRL-TK was also transfected as an internal control for transfection efficiency. 24 h after transfection, cells were serum-starved and treated with either SB203580 (SB, 20 µM) or Me2SO (DMSO) for another 24 h. Cells were harvested and assayed for luciferase activity. The average of four independent experiments is shown. Error bars denote the standard error of the mean. Fold activation was determined as luciferase activity relative to the vector control. B, 293 cells were transfected with Gal-Sap, Gal-lu, and the same group of plasmids used for panel A. Cells were treated with either SB203580 (20 µM) or Me2SO as for panel A, and lysates were assayed for luciferase activity. The average of four independent experiments is shown. Error bars denote the standard error of the mean. Fold activation was determined as for panel A.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GPCRs for a wide variety of ligands, including adrenergic hormones, neurotransmitters, autocoids, and peptide hormones, activate p38 MAPKs. Activation of p38 by GPCRs and heterotrimeric G proteins occurs by a number of mechanisms, but no single mechanism has been unequivocally coupled to a single receptor type. For example, in human embryonal kidney 293 cells, p38 is reportedly activated through M1 muscarinic and {beta}-adrenergic receptors, apparently through G{beta}{gamma} and G{alpha}q/11 (32). In adult rat ventricular myocytes, activation of p38 by Gi plays a protective role in {beta}-adrenergic receptor-stimulated apoptosis (33). In primary cultures of cardiac myocytes, activation of p38 by endothelin-1 involves neither Gi nor Go, but is probably controlled by Gq/11 acting through protein kinase C (34). In aggregate, the {alpha} subunits of Gs, Gq/11, Gi, G12, G13, and Go can each lead to activation of multiple MAPK family members, including p38, as can G{beta}{gamma} subunits (3539).

As noted earlier, the MAP3Ks used by GPCRs and G protein subunits to regulate p38 have thus far remained undefined. Here we show that M2 muscarinic receptors and G{alpha}o require TAOs and MEK3/6 as the primary intermediates activating p38 MAPK in 293 cells. Preliminary results suggest that this is the case in more than one cell type. Thus, we can now propose that the TAO family of MAP3Ks mediates signaling from physiological agonists such as carbachol to stress-responsive p38 MAPKs. Our findings also hint at weak stimulation of TAO2 by insulin. Although we have not yet been able to define conditions to demonstrate more significant activation of TAO by insulin, it is nevertheless possible that TAOs may participate in regulation of p38 not only by certain GPCRs but also by other hormones such as insulin with distinct signal transduction mechanisms.

The mechanism of regulation of TAO has not yet been determined. Earlier studies suggested that truncation of the C-terminal domains of TAO1 and TAO2 increased their activities, but no autoinhibitory regions have been identified (1, 2). The modest decrease in electrophoretic mobility of TAOs that occurs upon stimulation suggests that phosphorylation is involved in their mechanism of activation. Although the C terminus contains autophosphorylation sites (2), no regulatory sites have been identified. We have also been unable to activate purified TAO proteins with heterotrimeric G protein subunits in vitro, nor have we been able to coimmunoprecipitate endogenous TAO2 with G{alpha}o (data not shown). These results are most consistent with the likelihood that TAOs themselves are not the direct targets of G proteins, but rather that additional components, perhaps including a MAP4K, transmit the signal from G proteins to TAOs. Efforts are underway to define the mechanism of TAO activation and to find the molecules that link Go to TAOs.

Exploring downstream signaling, we found that TAO2 stimulates the phosphorylation of Elk1 on the major activating site and the transactivation of Elk1 and Sap1 in transfected cells. Comparable results have also been found for TAO1. Little or no effect of TAO2 was observed on promoters regulated by several other transcriptional activators, including NF-{kappa}B and AP-1, nor was a serum response element itself activated by TAO2 (data not shown). A component of AP-1, ATF2, is a well documented p38 substrate (40), yet activation of AP-1 driven luciferase was enhanced only ~2-fold by TAO2 in 293 cells. Regulation of AP-1 occurs at multiple levels; therefore, cooperative actions of the TAO-MEK-p38 pathway and other pathways may be required for the more robust activation of AP-1, consistent with the weak ability of constitutively active MEK6 to activate AP-1 (data not shown).

The capacity of TAO2 to stimulate transactivation of TCFs depends primarily on p38 but not ERK1/2. However, we cannot rule out a role for JNK/SAPK in connecting TAO to Elk1. The effects of TAO2 on Sap1 were more sensitive to inhibition by SB203580 than were effects on Elk1, although activation of neither was completely blocked by the inhibitor. One possible explanation is that part of the TAO2 effect on Elk1 is mediated by JNK/SAPK, perhaps through the activation of JNK/SAPK by MEK6. A second explanation is suggested by the specificity of SB203580. Four p38 isoforms have been identified, each of which may be regulated by distinct upstream pathways. For example, MEK6 is thought to be a common activator of p38{alpha}, {beta}, {gamma}, and {delta}, whereas MEK3 apparently only activates {alpha}, {gamma}, and {delta} isoforms (41, 42). SB203580 was used at 20 µM in the studies reported here, because a higher concentration inhibited effects of Raf BXB; a lower concentration was insufficient to block effects of MEK6DD (data not shown). Of the four known p38 isoforms, p38 {alpha} and {beta} are sensitive to SB203580, whereas {gamma} and {delta} are not (40). TAO- and MEK6-dependent transactivation of TCFs may, therefore, be mediated in part by the inhibitor-insensitive p38 isoforms. Future studies are directed toward determining how many of the p38 isoforms are regulated by TAOs in response to physiological ligands.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM53032 and GM34497 (to M. H. C. and A. G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} This work was performed in partial fulfillment of the requirements for a Ph.D. degree. Back

§ Present address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Back

Present address: Hyseq Pharmaceuticals, 670 Almanor Ave., Sunnyvale, CA 94085. Back

|| To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-3627; Fax: 214-648-3811; E-mail: mcobb{at}mednet.swmed.edu.

1 The abbreviations used are: TAO, thousand-and-one amino acids; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; MAP3K, MAPK kinase kinase; MEK6DD, constitutively active mutant of MEK 6; Raf BXB, constitutively active mutant of Raf-1; TCF, ternary complex factor; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; JIK, JNK/SAPK-inhibitory kinase; GPCR, gene protein-coupled receptor; TK, thymidine kinase; AP-1, activator protein 1; GST, glutathione S-transferase; ATF2, activating transcription factor 2. Back


    ACKNOWLEDGMENTS
 
We thank Bing-e Xu, Tara Beers Gibson, and Gray Pearson (University of Texas Southwestern Medical Center) for critical reading of the manuscript, Signal Pharmaceuticals for the MEK6 cDNA, Kunliang Guan for the MEK3 cDNA, members of the labs for proteins and constructs, Linda Hannigan and Jeff Laidlaw for technical assistance, and Dionne Ware for administrative assistance.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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