Regulation of Stress-responsive Mitogen-activated Protein (MAP) Kinase Pathways by TAO2

Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances. Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances. c-Jun N-terminal kinase mitogen-activated protein MAP/ERK kinases extracellular signal-regulated protein kinase c-Jun N-terminal kinases/stress-activated protein kinases MEK kinase polyacrylamide gel electrophoresis hemagglutinin TAO1 and TAO2 are closely related protein kinases whose cDNAs were originally isolated from rat based on sequence similarity to the yeast p21-activated protein kinase Ste20p (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The domain organization and regulation of TAOs is distinct from yeast p21-activated protein kinases. The kinase domain is at the TAO N terminus, and neither TAO1 nor TAO2 contains consensus motifs for activation by small G proteins. Each TAO is greater than a thousand amino acids in length; thus, each contains a regulatory domain of over 700 amino acids. A kinase that is over 90% identical to TAO2 was also identified in a screen for RNAs overexpressed in human prostate carcinoma; in this context it was named PSK for prostate-derived STE20-like kinase (3Moore T.M. Garg R. Johnson C. Coptcoat M.J. Ridley A.J. Morris J.D. J. Biol. Chem. 2000; 275: 4311-4322Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A third kinase, named JIK for JNK1 inhibitory kinase, has a similar organization and size and is over 60% identical to TAOs (4Tassi E. Biesova Z. Di Fiore P.P. Gutkind J.S. Wong W.T. J. Biol. Chem. 1999; 274: 33287-33295Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A chicken TAO-like kinase has also been found and is called KFC for kinase from chicken (5Yustein J.T. Li D. Robinson D. Kung H.J. Oncogene. 2000; 19: 710-718Crossref PubMed Scopus (14) Google Scholar). TAO1 and TAO2 activate stress-sensitive MAP kinase cascades in vitro by phosphorylating the upstream MAP/ERK kinases (MEKs), MEKs 3, 4, 6, and 7 (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Phosphorylation by TAOs increases their activities toward the downstream MAP kinases, p38 MAP kinase, and the c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs). These in vitro activities of TAO1 and TAO2 suggest that they function in stress responsive pathways within the cell as MEKK-level kinases. In vitro studies revealed a MEK binding domain just C-terminal to the TAO catalytic domain (2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This domain binds MEK3 and MEK6 with selectivity over other MEK family members; these MEKs are directed toward p38 family members. Both prostate-derived STE20-like kinase and kinase from chicken were reported to activate JNK/SAPKs in cotransfected cells (3Moore T.M. Garg R. Johnson C. Coptcoat M.J. Ridley A.J. Morris J.D. J. Biol. Chem. 2000; 275: 4311-4322Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Yustein J.T. Li D. Robinson D. Kung H.J. Oncogene. 2000; 19: 710-718Crossref PubMed Scopus (14) Google Scholar). JNK inhibitory kinase, in contrast, was reported to inhibit JNK/SAPK activity (4Tassi E. Biesova Z. Di Fiore P.P. Gutkind J.S. Wong W.T. J. Biol. Chem. 1999; 274: 33287-33295Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Effects of these TAO-related kinases on p38 were not reported. Numerous MEKK-level kinases have been implicated in regulating the stress-responsive JNK/SAPK and p38 pathways (reviewed in 6, 7). We wished to determine the behavior of TAOs in cells to clarify their roles in regulating these pathways. Thus, TAO2 was coexpressed in cells with various MEKs and MAP kinases, and endogenous MAPKs were immunoprecipitated from TAO2-transfected cells to examine effects of TAO2 expression on their activities. We also developed antibodies that could immunoprecipitate native TAO2 to determine whether TAO2 and MEKs could be coimmunoprecipitated and tested the capacities of various stimuli to activate TAO2. Finally, we examined the relationship between TAO2, p38, and JNK/SAPK activities during differentiation of C2C12 myoblasts. A fragment encoding TAO2-(1–451) was amplified by polymerase chain reaction with aBamHI site and EcoRI site incorporated at its 5′ and 3′ ends, respectively. The BamHI- andEcoRI-digested polymerase chain reaction product was ligated into BamHI- and EcoRI-digested pGEX-KG to create the pGEX-KG-TAO2-(1–451) plasmid. ABamHI/SalI-digested fragment from pGEX-KG-TAO2-(1–451) was ligated toBglII/SalI-digested pCMV5-Myc orBglII/SalI-digested pCMV5-HA to create pCMV5-Myc-TAO2-(1–451) or pCMV5-HA-TAO2-(1–451), respectively. The resulting plasmids were analyzed by EcoRI digestion and confirmed by sequencing. The D169A mutation was introduced into both plasmids with the QuikChange kit (Stratagene) according to the manufacturer's recommendations. The K82M and K64M mutations were created in the original pNPT7–5-MEK6 and pNPT7–5-MEK3 plasmids (kindly provided by Signal Pharmaceuticals and Kunliang Guan, respectively) also using the QuikChange kit. Escherichia coli strain BL21-DE3 transformed with either His6-MEK6KM or His6-MEK3KM was induced with 100 μm isopropyl-1-thio-β-d-galactopyranoside after growth to an outer diameter of 0.8 and grown at room temperature for another 7 h before harvesting. Purification procedures were as described (8Robbins D.J. Zhen E. Owaki H. Vanderbilt C. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar). MEK4KM and SAPKβKA protein preparations were kindly provided by Mahesh Karandikar. ATF2-(1–254) and Myc-(1–103) protein preparations were kindly provided by Don Arnette. pCEP4-HA-ERK2, pSRα-HA-JNK1, pSRα-HA-SAPKβ, and pCEP4-HA-p38α were as described (9English J.M. Pearson G. Hockenberry T. Shivakumar L. White M.A. Cobb M.H. J. Biol. Chem. 1999; 274: 31588-31592Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar). pCMV5-HA-MEK1, pCMV5-HA-MEK4, pCMV5-Myc-MEK3, pSRα-HA-MEK6, pCMV5-MEKK1, and RasVal12 were kindly provided by Jeff Frost and Jennifer Swantek and were described elsewhere (11Swantek J.L. Christerson L. Cobb M.H. J. Biol. Chem. 1999; 274: 11667-11671Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar, 13Frost J.A. Steen H. Shapiro P.S. Lewis R. Ahn J. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). pCS3+-MT-Myc-MEK7 was provided by Shuichan Xu. 293 cells and Neuro2A cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% l-glutamine, and 100 units/ml penicillin/streptomycin. ATT20, GC-B6, PC12, and PC12M were cultured under similar conditions. C2C12 cells were purchased from ATCC. To prepare whole cell lysates, cells were washed once with cold phosphate-buffered saline and lysed with Triton lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 80 mm β-glycerophosphate, 0.5 mmsodium orthovanadate, 1 mm EDTA, 20 μg/ml aprotinin, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride). Insoluble material was pelleted by centrifugation, and supernatants were removed, snap frozen in liquid nitrogen, and stored at −80° C. For cell fractionation, cells were washed once with phosphate-buffered saline and resuspended in hypotonic buffer (10 mm Hepes, pH 7.6, 1.5 mmMgCl2, 10 mm NaCl, 1 mm EDTA, 1 mm EGTA, and supplemented with protease inhibitors and phosphatase inhibitors as above). Cells were lysed with a Dounce apparatus, and the nuclei were collected by sedimentation at 800 × g for 5 min. A particulate fraction was collected by sedimenting the supernatant at 100,000 × g for 30 min. The nuclear pellet was washed with 20 mm Hepes, pH 7.6, 2.5% glycerol, 0.42 m NaCl, 1.5 mmMgCl2, 1 mm EDTA, and 1 mm EGTA for 30 min. Both the washed nuclear pellet and particulate fraction were lysed with 1% SDS, 10 mm Tris-HCl, pH 7.6, 1 mm EDTA, 1 mm EGTA, and supplemented with protease inhibitors and phosphatase inhibitors as above. Three μg of various MEK or MAPK constructs were cotransfected with 2 μg of either empty vector control or TAO2 constructs in 293 cells grown to 80% confluence on 60-mm dishes using the calcium phosphate method. Cells were serum-starved for 24 h beginning the second day and then harvested. Alternatively, cells were transfected with 5 μg of either empty vector control or TAO2 constructs to examine the activities of endogenous MAPKs. A polyclonal antiserum (U2253) was raised against the antigenic peptide MPAGGRAGSLKDPDVAELFFK (residues 1–21 of TAO2 protein) in rabbits. This peptide was conjugated to Limulus hemocyanin (14Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (282) Google Scholar) and dialyzed into phosphate-buffered saline. A total of five boosts were performed. The final bleed of U2253 was used in all subsequent studies involving TAO2 antibodies. For immunoblot analysis, recombinant proteins or cell lysates were subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in TBST (20 mm Tris, pH 8.0, 500 mm NaCl, and 0.05% Tween20) overnight and then incubated with primary antibody diluted in TBST plus 0.5% milk at a 1:10,000 dilution for 1 h. After three washes with TBST, the membranes were incubated with secondary antibody diluted in TBST plus 0.5% milk for 30 min. Membranes were washed again with TBST three times and Tris-buffered saline once and then developed using and were used to immunoprecipitate and to immunoblot endogenous (14Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (282) Google Scholar). J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar) or was used to immunoprecipitate or to immunoblot endogenous p38, respectively. both and of p38. JNK/SAPK and MEK6 were immunoprecipitated and with and respectively. was also purchased from antibody and antibody were from and the Cell respectively. MEKs or MAPKs were immunoprecipitated from of whole cell protein the antibodies and 30 of protein were washed three times with 1 of 500 and 20 mm Tris-HCl, pH washed one with 1 of 20 mm Tris, pH 8.0, and 10 mmMgCl2, and subjected to in vitro kinase (2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). TAO2 was immunoprecipitated from 1.5 protein as described in M. S. Cobb M.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) with 5 of TAO2 antibody (U2253) and 30 of protein were washed three times with 1 of mm NaCl, 20 mm Tris-HCl, pH and Triton X-100, washed one with 1 of 20 pH 8.0, and 10 mm and subjected vitro kinase were as that the was 10 were as the of the in the in the and the control Phosphorylation of was by liquid C2C12 cells were purchased from ATCC. Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine to 80% confluence and then in Dulbecco's modified Eagle's medium with for to differentiation as described Z. K. M. Mol. Cell. Biol. 2000; PubMed Scopus Google A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of this medium was with medium cells were with either or μm in the cells were in lysis buffer as and were isolated and as described in the To TAO2 selectivity in experiments were performed. In a an MAPK was transfected into 293 cells with either the empty vector control or with a TAO2 The overexpressed MAPK was immunoprecipitated from the cells and using substrates cotransfected with various TAO2 toward its 1 was activated by the TAO2 constructs toward 1 The activity of on its was also by cotransfected or TAO2-(1–451) 1 In to these was not activated toward its Myc-(1–103) by of the cotransfected TAO2 plasmids the that TAO2 is not an upstream of the TAO2-(1–451) to be than or is studies with protein in suggested that it contains a regulatory domain that its activity (2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). on kinase from chicken also an inhibitory of the on its catalytic domain (5Yustein J.T. Li D. Robinson D. Kung H.J. Oncogene. 2000; 19: 710-718Crossref PubMed Scopus (14) Google Scholar). The in between and TAO2-(1–451) is to the and of the binding This domain of TAO2 has been to (2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Thus, the TAO2 catalytic the binding site thus, selectivity stress-sensitive The studies demonstrated that JNK/SAPK and p38 be activated by TAO2 in we examined effects of TAO2 on endogenous MAPKs. vector or kinase-dead were transfected into 293 and endogenous p38, JNK/SAPK, and were immunoprecipitated from transfected cells and using Both endogenous p38 and endogenous JNK/SAPK were activated by TAO2-(1–451) toward their and the activity of endogenous was not to an The TAO2 to its expression not to activate stress-sensitive MAPKs in similar TAO1 also to or activate in cells (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). the kinase activity of TAO2 be by or by not A was also to examine of TAO2 for downstream The overexpressed MEK proteins were immunoprecipitated from the cells and using the MAPK as The MAPK substrates were in to RasVal12 was as a control for and was as a control for and of toward its was not by cotransfected TAO2 3 A, nor were and activated by TAO2 toward their SAPKβKA 5 and In activities of MEK3 and MEK6 toward p38 were by cotransfected TAO2 and that TAO2 selectively activates MEK3 and MEK6 of the p38 in A kinase-dead to activate cotransfected MEK6 that TAO2 kinase activity is pCMV5-Myc-TAO2-(1–451) and were transfected into 293 cells TAO2 proteins were then immunoprecipitated from the cells their and using MEKs 3, 4, and 6 as were on SDS-PAGE and subjected to and were by TAO2-(1–451) to 1 and the TAO2-(1–451) was by TAO2 was to of the consistent with its of catalytic activity 4, The of the immunoprecipitated protein to under these is a of its for to MEK3 and A polyclonal TAO2 antiserum was against an N-terminal peptide from TAO2. The antibody was with antigenic peptide to that the peptide blocked recognition of the TAO2 We examined cell of TAO2 is in (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). the we found that TAO2 is in cell 293 5 TAO2 is found in both and particulate of 293 cells but is not in nuclei 5 the of the binding site for TAO2 we endogenous TAO2 associates with 293 cells were transfected with TAO2 was immunoprecipitated from the cells with the antibody and with was in the TAO2 To whether be demonstrated with both endogenous TAO2 was immunoprecipitated from 293 cells with the and the were then with a MEK6 was found in with endogenous TAO2 but not with the MEKK-level of the 6 that this is could be between TAO2 and or not consistent with and in studies (2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). We wished to determine whether TAO2 itself be activated by stress stimuli that activate JNK/SAPK or p38. To this 293 cells were serum-starved for 24 h and then to NaCl, or the and TAO2 contains of is an to its activity (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). TAO2 was immunoprecipitated from the cells and its ability to was TAO2 was by and by NaCl, and nocodazole 7 TAO2 immunoprecipitated from or cells was then using kinase-dead MEK3 and MEK6 as substrates to that is a of TAO2 activity under these circumstances. Phosphorylation of both MEK3 and MEK6 by TAO2 was by Thus, and to a lesser other cell increased TAO2 C2C12 myoblasts be induced to and in Z. K. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. M. J. Cell. PubMed Scopus Google Scholar). p38 is activated during the differentiation and its activation is for differentiation induced by either or growth In contrast, JNK/SAPK activities have been reported to and is by is during differentiation consistent with of JNK/SAPKs in differentiation Z. K. M. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. M. J. Cell. PubMed Scopus Google Scholar). We wished to determine whether in TAO2 activity during that the idea that activation of endogenous TAO2 activation of p38 but not the JNK/SAPK C2C12 cells were in differentiation medium with or the p38 for were in the day in the of but were cells been to The of p38 protein not as its activity increased on or before day 2 and day and the The of TAO2 protein in the cells over the differentiation in a or to activity increased over the of differentiation also in an the in its The of JNK/SAPK protein in the cells its activity over the In to p38 and TAO2, JNK/SAPK activity in cells on day was to the effects of growth JNK/SAPK activity is not for its activation be during this Thus, TAO2 activity p38 activity but not JNK/SAPK This is consistent with the idea that TAO2 is to p38 but not JNK/SAPKs in studies have revealed the of a of MEKK-level kinases in TAO1 and TAO2 (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, A. D. EMBO J. 1996; PubMed Scopus Google Scholar, K. K. H. K. T. E. K. PubMed Scopus Google Scholar, K. Z. 1998; PubMed Scopus Google Scholar). This for activation of MAP kinase in a and cell In MAPK regulation by MEKK-level kinases with the ability of to and activate MEK proteins in MAPK Thus, we examined the of TAOs in vitro in this TAO2 in cell In the catalytic of TAO1 and TAO2 activated MEKs 3, 4, and 6 toward their p38 and JNK/SAPK (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In contrast, an TAO2 was cotransfected with various MEK proteins MEK3 and MEK6 of the p38 were TAO2 activated both cotransfected and endogenous p38 and TAO2, p38, and JNK/SAPK activities during C2C12 we were to that that to activation of endogenous TAO2 are with increases in p38 but not JNK/SAPK This of the that TAO proteins are to the p38 and have in regulating The for MEKs 3 and 6 the of the MEK binding domain of TAO2. we identified this TAO domain C-terminal to its kinase domain and found that it an N-terminal in MEKs 3 and 6 (1Hutchison M. Berman K. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Chen Z. Hutchison M. Cobb M.H. J. Biol. Chem. 1999; 274: 28803-28807Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). we have not found a site for p38 binding on TAO2. the JNK/SAPK and neither TAO2 nor are activated by TAO2 in activation of JNK/SAPK by TAO be in to TAO2 and to the of vitro kinase studies have that MEK6 JNK/SAPK to an extent, the activity is than that of toward JNK/SAPK not have in of for the of these from studies of protein kinase and the and JNK/SAPK MAPK T. J.D. 1997; PubMed Scopus Google Scholar, T. M. T. E. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, J.L. T. Cobb M.H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. A.J. J. M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). A site for p38 in MEK6 and an of to to of p38 H. EMBO J. 2000; 19: PubMed Scopus Google Scholar). The of TAO with and p38 the MEK in for their in the of their physiological selectively with and phosphorylating MEK3 and MEK6 in TAO2 in to these upstream of p38, from TAO2 to and to p38. implicated in regulating JNK/SAPK and p38 are responsive to For be activated by of or M. S. Cobb M.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, T. E. A. Johnson Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, T. S. Johnson J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and is to and K. K. H. K. S. T. K. E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). To stimuli or that TAO2, we a TAO2 antibody that to immunoprecipitate endogenous TAO2 proteins from cells with a of the than stimuli on TAO2 activity not The as as other activated TAO2 that TAO2 be in stress-responsive pathways. TAO2 not activation of p38 by not are activated by and all to p38 this is it be to the of these to p38 we have been to that kinase-dead TAO2 was in activation of p38 by L. A. G. and M. in Thus, the of studies to a of regulating the p38 MAP kinase We and Pearson for of the Signal Pharmaceuticals for the MEK6 Kunliang for the MEK3 of the Cobb for proteins and and for