Modulation of F-actin Rearrangement by the Cyclic AMP/cAMP-dependent Protein Kinase (PKA) Pathway Is Mediated by MAPK-activated Protein Kinase 5 and Requires PKA-induced Nuclear Export of MK5
69
Citation
60
Reference
10
Related Paper
Citation Trend
Abstract:
The MAPK-activated protein kinases belong to the Ca2+/calmodulin-dependent protein kinases. Within this group, MK2, MK3, and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified, and the only known biological role is in ras-induced senescence and in tumor suppression. Here we demonstrate that activation of cAMP-dependent protein kinase (PKA) or ectopic expression of the catalytic subunit Calpha in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Calpha and MK5 and the ability of Calpha to enter the nucleus. Calpha and MK5, but not MK2, interact in vivo, and Calpha increases the kinase activity of MK5. Moreover, Calpha augments MK5 phosphorylation, but not MK2, whereas MK5 does not seem to phosphorylate Calpha. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that small interfering RNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangement. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA-provoked F-actin remodeling. Our results describe a novel interaction between the PKA pathway and MAPK signaling cascades and suggest that MK5, but not MK2, is implicated in PKA-induced microfilament rearrangement.Keywords:
ASK1
The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.
Protein kinase R
Cyclin-dependent kinase 9
ASK1
EIF-2 kinase
MAP2K7
c-Raf
RNA Silencing
Cite
Citations (103)
Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKcooH) binds to GST-Rasv12 in a GTP-dependent manner. Purified bacterially expressed MEKKcooH binds to GST-Rasv12(GTPγS) (GTPγS is guanosine 5’-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKcooH to GST-Rasv12(GTPγS). MEKKcooH complexed with GST-Rasv12(GTPγS) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras•GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families. Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKcooH) binds to GST-Rasv12 in a GTP-dependent manner. Purified bacterially expressed MEKKcooH binds to GST-Rasv12(GTPγS) (GTPγS is guanosine 5’-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKcooH to GST-Rasv12(GTPγS). MEKKcooH complexed with GST-Rasv12(GTPγS) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras•GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.
ASK1
Cyclin-dependent kinase 9
MAP2K7
c-Raf
Cyclin-dependent kinase 4
Cite
Citations (178)
MAP2K7
ASK1
Cyclin-dependent kinase 9
MAPK14
c-Raf
Protein kinase R
Cyclin-dependent kinase 4
Cite
Citations (0)
MAP2K7
Cyclin-dependent kinase 9
ASK1
Cyclin-dependent kinase 4
c-Raf
Cite
Citations (149)
MAP2K7
ASK1
Cyclin-dependent kinase 9
MAPK14
Protein kinase R
c-Raf
Cyclin-dependent kinase 4
Cite
Citations (10)
Xenopus 45-kDa mitogen-activated protein (MAP) kinase kinase (MAPKK) is a serine/threonine/tyrosine kinase, which activates MAP kinase (MAPK) by phosphorylating its threonine and tyrosine residues. MAPKK is active only when its threonine and/or serine residues are phosphorylated. We have identified from Xenopus eggs two protein kinases responsible for phosphorylation of MAPKK. The two kinases are separated by Sephacryl S-300 gel filtration chromatography. The higher molecular weight kinase phosphorylates MAPKK previously dephosphorylated and inactivated by phosphatase 2A treatment on mainly serine and slightly threonine residues, and reactivates the MAPKK, and is thus assumed to work as MAPKK kinase (MAPKKK) in vivo. The lower molecular weight kinase, identified as MAPK, phosphorylates the dephosphorylated MAPKK on mainly threonine and faintly serine residues, but does not reactivate the MAPKK activity. As Xenopus MAPKK contains a single phosphorylation consensus sequence (PXT388P) for MAPK in the C-terminal region, this T388 residue may be a major phosphorylation site catalyzed by MAPK. Thus, Xenopus MAPKK is phosphorylated in mature oocytes by not only an upstream kinase, MAPKKK, but also a downstream kinase, MAPK.
Cyclin-dependent kinase 9
MAP2K7
ASK1
MAPK14
MAPK7
Cyclin-dependent kinase 4
MAPKAPK2
c-Raf
Protein kinase R
Cite
Citations (128)
ABSTRACTAgents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression. ACKNOWLEDGMENTSWe thank P. Worley and H. Mischak for the c-Raf kinase expression vectors, M. Karin for HA-tagged MAP kinase, and S. Taylor for PKI and Kemptide.This work was supported in part by NSF grant MCB-9506327 and a University of California Cancer Research Coordinating Committee grant to R.B.P. and USPHS grant GM4960 and an American Heart Association grant to G.R.B. The Bio-Rad MRC-1024 laser scanning confocal system is part of the San Diego Microscopy and Imaging Resource at UCSD supported by NIH grant RR04050 (principal investigator, M. H. Ellisman); we thank N. Alinejad for technical assistance.
Cyclin-dependent kinase 9
MAP2K7
ASK1
Cyclin-dependent kinase 4
c-Raf
cGMP-dependent protein kinase
Protein kinase R
MAPK14
Cite
Citations (109)
A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser189, comparable to Thr183, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members. A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser189, comparable to Thr183, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.
MAP2K7
ASK1
c-Raf
Cyclin-dependent kinase 9
MAPK14
Cyclin-dependent kinase 4
Cite
Citations (43)
The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Δ tos3Δ elm1Δ yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-β-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells. The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Δ tos3Δ elm1Δ yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-β-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells. The Snf1/AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, AMP-activated protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; JNK, c-Jun N-terminal kinase; IKK, IκB kinase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; IL-1, interleukin-1; GAD, Gal4 activation domain; HA, hemagglutinin; GST, glutathione-S-transferase; GFP, green fluorescent protein; SC, synthetic complete medium; WT, wild type; KD, kinase domain. family has major roles in regulation of glucose and lipid metabolism, maintenance of cellular energy homeostasis, and cellular stress responses (reviewed in Refs. 1Kemp B.E. Stapleton D. Campbell D.J. Chen Z.P. Murthy S. Walter M. Gupta A. Adams J.J. Katsis F. Van Denderen B. Jennings I.G. Iseli T. Michell B.J. Witters L.A. Biochem. Soc. Trans. 2003; 31: 162-168Crossref PubMed Google Scholar and 2Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2372) Google Scholar). In mammalian cells, reduced energy availability (high cellular AMP:ATP ratio) causes activation of AMPK, which promotes glucose transport and ATP-generating metabolic processes, inhibits ATP-consuming processes, and regulates transcription. AMPK is also regulated by leptin, adiponectin, and ghrelin (3Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1709) Google Scholar, 4Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3537) Google Scholar, 5Andersson U. Filipsson K. Abbott C.R. Woods A. Smith K. Bloom S.R. Carling D. Small C.J. J. Biol. Chem. 2004; 279: 12005-12008Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar) and has a role in controlling appetite and food intake (5Andersson U. Filipsson K. Abbott C.R. Woods A. Smith K. Bloom S.R. Carling D. Small C.J. J. Biol. Chem. 2004; 279: 12005-12008Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 6Minokoshi Y. Alquier T. Furukawa N. Kim Y.B. Lee A. Xue B. Mu J. Foufelle F. Ferre P. Birnbaum M.J. Stuck B.J. Kahn B.B. Nature. 2004; 428: 569-574Crossref PubMed Scopus (1361) Google Scholar). In humans, AMPK is an important therapeutic target for type 2 diabetes (2Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2372) Google Scholar, 7Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar). In the yeast Saccharomyces cerevisiae, Snf1 protein kinase (8Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (539) Google Scholar) is the ortholog of AMPK (9Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 10Mitchelhill K.I. Stapleton D. Gao G. House C. Michell B. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1994; 269: 2361-2364Abstract Full Text PDF PubMed Google Scholar). Snf1 protein kinase, like AMPK, is heterotrimeric, comprising a catalytic subunit (Snf1/α), and two regulatory subunits (β and Snf4/γ). Mutation of SNF1 causes the Snf– (sucrose-nonfermenting) phenotype, which is characterized by inability to utilize carbon sources that are less preferred than glucose. Like AMPK, Snf1 protein kinase regulates transcription, metabolic enzymes, and transporters in response to stress, particularly carbon stress (reviewed in Refs. 11Carlson M. Curr. Opin. Microbiol. 1999; 2: 202-207Crossref PubMed Scopus (466) Google Scholar and 12Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1288) Google Scholar). The Snf1/AMPK-activating kinases in the kinase cascade are also highly conserved between yeast and mammals (Fig. 1). Yeast contains three homologous kinases that phosphorylate the activation loop Thr-210 of the Snf1 catalytic subunit: Sak1 (Snf1-activating kinase, previously Pak1), Tos3, and Elm1 (13Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 14Nath N. McCartney R.R. Schmidt M.C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (126) Google Scholar, 15Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Mutant yeast cells lacking these three kinases (sak1Δ tos3Δ elm1Δ cells) cannot activate Snf1 and exhibit a Snf– phenotype (13Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 15Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Their mammalian sequence homologs, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), phosphorylate Thr-172 in the activation loop of the AMPK catalytic subunit and activate AMPK in vitro and in vivo (13Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 17Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 18Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1480) Google Scholar, 19Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, 20Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar, 21Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar, 22Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). The yeast and mammalian kinases exhibit striking functional interchangeability. Tos3 and Elm1 phosphorylate and activate AMPK in vitro (13Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar, 15Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). LKB1, in complex with its partners STRADα and MO25α, and CaMKK phosphorylate Snf1 in vitro and activate Snf1 in sak1Δ tos3Δ elm1Δ mutant yeast, conferring a Snf+ growth phenotype (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). Here, we took advantage of this conservation of the Snf1/AMPK pathway and exploited the yeast genetic system in an effort to identify new members of the AMPK kinase family. Given that yeast, a simple unicellular organism, has three Snf1 protein kinase kinases, it seems likely that mammals have multiple AMPK kinases. The heterologous function of LKB1 and CaMKK in yeast provides the basis for a convenient and powerful genetic selection for mammalian AMPK kinases: the restoration of the Snf+ growth phenotype in sak1Δ tos3Δ elm1Δ mutant yeast. The power of this selection lies not only in its simplicity but also in its sensitivity. The Snf1 pathway is robust, and very little activity is required for growth; for example, expression of LKB1 alone restores growth despite causing only a modest elevation of Snf1 catalytic activity (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This sensitivity is important because the catalytic subunit of an AMPK kinase must function in yeast without other mammalian proteins, either alone or in association with yeast orthologs. We have transformed sak1Δ tos3Δ elm1Δ yeast cells with a mouse embryo cDNA library in a yeast expression vector and selected for cDNAs that confer the Snf+ growth phenotype. This genetic selection yielded clones encoding two authentic AMPK kinases, LKB1 and CaMKKβ, and also transforming growth factor β (TGF-β)-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. TAK1 was identified as a mediator of TGF-β signaling in mammalian cells (23Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1187) Google Scholar) and was shown to participate in bone morphogenetic protein signaling in Xenopus development (24Shibuya H. Iwata H. Masuyama N. Gotoh Y. Yamaguchi K. Irie K. Matsumoto K. Nishida E. Ueno N. EMBO J. 1998; 17: 1019-1028Crossref PubMed Scopus (192) Google Scholar). TAK1 is also activated by the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) and by bacterial lipopolysaccharide and regulates IκB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 MAPK pathways (25Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 26Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 27Lee J. Mira-Arbibe L. Ulevitch R.J. J. Leukocyte Biol. 2000; 68: 909-915Crossref PubMed Google Scholar, 28Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 29Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1682) Google Scholar, 30Takaesu G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. Matsumoto K. Gaynor R.B. J. Mol. Biol. 2003; 326: 105-115Crossref PubMed Scopus (328) Google Scholar). TAK1 phosphorylates and activates IKK, MKK4, MKK3, and MKK6 (23Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1187) Google Scholar, 25Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 26Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 29Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1682) Google Scholar, 31Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 32Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Three TAK1-binding proteins have been identified; TAB1 increases TAK1 catalytic activity (33Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar), and TAB2 and TAB3 function as adaptors in cytokine signaling pathways (28Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 34Ishitani T. Takaesu G. Ninomiya-Tsuji J. Shibuya H. Gaynor R.B. Matsumoto K. EMBO J. 2003; 22: 6277-6288Crossref PubMed Scopus (220) Google Scholar, 35Cheung P.C. Nebreda A.R. Cohen P. Biochem. J. 2004; 378: 27-34Crossref PubMed Scopus (136) Google Scholar). We present genetic and biochemical evidence that TAK1 phosphorylates Thr-210 of Snf1 and functions as a Snf1-activating kinase. We further show that TAK1 phosphorylates Thr-172 of the AMPK catalytic subunit in vitro and that expression of TAK1 and TAB1 stimulates phosphorylation of AMPK in HeLa cells. These findings suggest that TAK1 is a member of the Snf1/AMPK kinase family. Yeast Strains—S. cerevisiae strains were W303-1A (MATa ura3 trp1 ade2 his3 can1 leu2), MCY4908 (W303-1A snf1Δ10), MCY5138 (MATα sak1Δ::kanMX4 tos3Δ::kanMX4 elm1Δ::ADE2 ura3 trp1 ade2 his3 can1 leu2), MCY5115 (MATα sak1Δ::kanMX4 ura3 trp1 ade2 his3 can1 leu2), and MCY5125 (W303-1A elm1Δ::kanMX4). Synthetic complete (SC) medium lacking appropriate supplements was used to select for plasmids. Selection for Mammalian Snf1-activating Kinases in Yeast—DNA of a two-hybrid library prepared from mouse 17-day embryo cDNAs in a yeast expression plasmid vector carrying the LEU2 marker (Clontech catalog number 638846) was used to transform (36Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2148) Google Scholar) yeast strain MCY5138 (see Fig. 2). A total of 5 × 106 transformants were selected on 500 plates of SC solid medium containing 2% glucose and lacking leucine. Colonies from each plate were resuspended in SC medium and transferred to a fresh plate of SC-leucine solid medium containing 2% raffinose plus the respiratory inhibitor antimycin A (1 μg/ml). Growth on this medium requires activation of Snf1 protein kinase; in control experiments, colonies expressing LKB1 appeared in 3–7 days. After 5–7 days, two colonies from each plate were picked and retested for growth. Plasmid DNAs were rescued by passage through bacteria, retested by transformation of MCY5138, and sequenced. One plasmid was saved from each plate. Plasmids—pK98, expressing GAD-TAK1, was recovered above. pRH104 (37Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (74) Google Scholar), pRH105 (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and pRH123 (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) express HA-Sak1, HA-Tos3, and HA-CaMKKα, respectively, from vector pWS93 (38Song W. Carlson M. EMBO J. 1998; 17: 5757-5765Crossref PubMed Scopus (104) Google Scholar). GAD-Snf1 was expressed from pSG1 (39Jiang R. Carlson M. Genes Dev. 1996; 10: 3105-3115Crossref PubMed Scopus (243) Google Scholar). pMM25 and pMM29 express HA-TAK1 and LexA-TAB1, respectively, from mouse cDNAs (Open Biosystems) cloned into pWS93 (38Song W. Carlson M. EMBO J. 1998; 17: 5757-5765Crossref PubMed Scopus (104) Google Scholar) and pBTM116 (40Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4939) Google Scholar). pMM26, expressing TAK1K63W with Lys-63 altered to Trp, was constructed from pMM25 by using the QuikChange site-directed mutagenesis kit (Stratagene); three independent mutant plasmids behaved similarly. cDNAs encoding residues 1–318 of the wild-type (WT) and mutant kinase domain of AMPK, AMPK-KD-WT, and AMPK-KD-T172A (gifts of L. Witters; see Ref. 41Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), were transferred to vector pET32a (Novagen) to yield pMM45 and pMM57, respectively, expressing His-tagged proteins. pMM33, pMM35, and pMM37 express TAK1, TAK1K63W, and TAB1, respectively, from vector pCMV-FLAG2 (Invitrogen); TAK1 proteins were not recognized by anti-FLAG, although sequence analysis confirmed the FLAG tag. Snf1T210A and Gal83, tagged with green fluorescent protein (GFP), were expressed from their native promoters on pKH43 (37Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (74) Google Scholar) and pRT13 (42Hedbacker K. Townley R. Carlson M. Mol. Cell. Biol. 2004; 24: 1836-1843Crossref PubMed Scopus (77) Google Scholar). Analysis of Proteins—Proteins were separated by SDS-PAGE in 8% polyacrylamide. Immunoblot analysis was carried out with anti-Snf1 (8Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (539) Google Scholar), monoclonal anti-HA (12CA5), anti-LexA (Invitrogen), anti-FLAG (Sigma), anti-TAK1 (Upstate), anti-phospho-Thr-172-AMPK and anti-AMPKα (Cell Signaling Technologies), and anti-His6-peroxidase (Roche Diagnostics). Antibodies were detected with chemiluminescence using ECL Plus or ECL Advance (Amersham Biosciences). Blots were incubated in 0.2 m glycine, pH 2, for 5 min and washed before reprobing. Assay of Snf1 Activity by Phosphorylation of SAMS Peptide—Yeast cells were grown to mid-log phase in SC medium containing 2% glucose, collected by filtration, incubated in SC with 0.05% glucose for 15 min, and collected by filtration. Extracts were prepared from two independent cultures, Snf1 was partially purified, and phosphorylation of the synthetic peptide HMRSAMSGLHLVKRR (SAMS peptide; Ref. 43Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (380) Google Scholar) was assayed as described (9Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 37Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (74) Google Scholar). Each preparation was assayed twice, with dilutions to confirm linearity. Kinase activity is expressed as nanomoles of phosphate incorporated into the peptide per minute per milligram of protein (43Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (380) Google Scholar). Assay of Phosphorylation of Recombinant Snf1 and AMPK Catalytic Domains—Glutathione S-transferase (GST) fusions to the mutant Snf1 catalytic domains Snf1KD-K84R and Snf1KD-T210A were expressed in bacteria and purified as described (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). His-tagged AMPK-KD-WT and AMPK-KD-T172A catalytic domains were expressed in bacteria and purified using AKTA fast protein liquid chromatography on chelating HiTrap resin (Amersham Biosciences). Bound proteins were eluted with a linear gradient as described by the manufacturer. Cultures of MCY5138 expressing HA-TAK1 and/or LexA-TAB1 were grown in SC with 2% glucose, collected by filtration, incubated in 0.05% glucose for 30 min, and collected by filtration. HA-tagged proteins were immunoprecipitated from extracts (200 μg) with anti-HA antibody as described (13Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (487) Google Scholar). Kinases were assayed for phosphorylation of GST-Snf1KD (3 μg) or AMPK-KD (0.5 μg) substrates using [γ-32P]ATP as described (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). His-tagged recombinant human TAK1-TAB1 fusion protein (100 ng; Upstate catalog number 14-600) was incubated with substrates and cold ATP. Analysis of Phosphorylation of AMPK in HeLa Cells—HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mm l-glutamine. Cells were transfected with DNAs (8 μg/6-cm dish) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. TNF-α and IL-1β were purchased from R&D Systems. Cells were lysed by the addition of ice-cold lysis buffer as described (20Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar), except without prior rinsing. Lysates were collected immediately and clarified by brief centrifugation in the cold. Genetic Selection for Mammalian Snf1-activating Kinases in Yeast—The sak1Δ tos3Δ elm1Δ mutant yeast strain lacks all three native Snf1 protein kinase kinases and therefore exhibits the Snf– (sucrose-nonfermenting) phenotype, which is characterized by the ability to utilize glucose but not alternative carbon sources. To identify mammalian Snf1-activating kinases, and thus candidates for AMPK kinases, we selected mammalian cDNAs that allow sak1Δ tos3Δ elm1Δ cells to grow on raffinose, as shown schematically in Fig. 2. We used a library of mouse 17-day embryo cDNAs fused to the Gal4 activation domain (GAD) in a yeast expression vector to transform sak1Δ tos3Δ elm1Δ yeast cells and then selected for growth on raffinose. We recovered the cDNA plasmid from Snf+ colonies by passage through Escherichia coli, retransformed sak1Δ tos3Δ elm1Δ yeast cells to confirm that the cDNA conferred a Snf+ phenotype, and identified the cDNA by sequencing. In a screen of 5 × 106 transformants, we recovered 49 cDNA clones expressing LKB1, five expressing CaMKKβ, and six expressing TAK1, also known as MAPKKK7 (23Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1187) Google Scholar). This selection also yielded seven cDNAs expressing transcription factors, which were not characterized further, but none expressing CaMKKα, which we previously showed to function in yeast (Ref. 16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar; see Fig. 3B). The recovery of LKB1 and CaMKKβ, which are both known AMPK kinases, validates this approach. TAK1 is thus a candidate for a Snf1-activating kinase and potentially an AMPK kinase. Growth Phenotype Conferred by TAK1 Requires Snf1 Protein Kinase—We first sought to confirm that the ability of TAK1 to confer growth on raffinose requires Snf1 protein kinase. A cDNA plasmid expressing GAD-TAK1 was used to transform snf1Δ mutant cells. The transformants did not grow on raffinose (Fig. 3A), indicating that TAK1 requires Snf1 protein kinase to confer a Snf+ phenotype and does not function by bypassing Snf1. In control experiments, expression of Snf1 in the mutant cells restored growth, as expected. The cDNA clones recovered from the library expressed TAK1 with GAD fused to its N terminus. To exclude the possibility that this fusion protein had aberrant function, we expressed full-length TAK1, tagged with a triple-HA epitope at its N terminus, from the yeast ADH1 promoter of vector pWS93. Expression of this HA-tagged TAK1 allowed sak1Δ tos3Δ elm1Δ cells to grow on raffinose (Fig. 3B) and on glycerol plus ethanol (Fig. 3C); HA-TAK1 was used in all subsequent experiments. TAB1, a TAK1-binding protein identified in the two-hybrid system, increases the catalytic activity of TAK1 (33Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar); however, TAK1 acts independently of TAB1 in some signaling pathways in mammalian cells (44Shim J.H. Xiao C. Paschal A.E. Bailey S.T. Rao P. Hayden M.S. Lee K.Y. Bussey C. Steckel M. Tanaka N. Yamada G. Akira S. Matsumoto K. Ghosh S. Genes Dev. 2005; 19: 2668-2681Crossref PubMed Scopus (603) Google Scholar). Coexpression of LexA-TAB1 from the ADH1 promoter did not improve growth of sak1Δ tos3Δ elm1Δ cells on raffinose (Fig. 3B), although some improvement was noted on glycerol-ethanol (Fig. 3C); expression was confirmed by immunoblot analysis (data not shown and Fig. 4C). In addition, TAK1, with or without TAB1, did not allow raffinose utilization by snf1Δ cells expressing mutant Snf1T210A with the activation loop Thr-210 replaced by Ala, as predicted if TAK1 functions by phosphorylating Thr-210 (data not shown). TAK1 Activates Snf1 Protein Kinase in Vivo—To determine whether TAK1 activates Snf1 protein kinase in vivo, we assayed Snf1 catalytic activity in sak1Δ tos3Δ elm1Δ mutant cells expressing HA-TAK1. Cells were grown to mid-log phase in glucose and then shifted to medium containing 0.05% glucose for 30 min, a condition that results in activation of Snf1 in wild-type cells. Cell extracts were prepared, and phosphorylation of a synthetic peptide substrate, the SAMS peptide, by partially purified Snf1 protein kinase was determined. The presence of HA-TAK1 in the mutant cells resulted in the activation of Snf1 to levels similar to those caused by CaMKKα (Fig. 4A), which is roughly 2-fold reduced relative to wild type (16Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Coexpression of LexA-TAB1 with HA-TAK1 did not substantially increase activation of Snf1 (Fig. 4A), consistent with the growth phenotypes (Fig. 3). Amounts of Snf1 protein were similar in all assays, and coexpression of TAB1 did not result in elevated levels of TAK1, although TAK1 appeared to stabilize TAB1, as judged by immunoblot analysis (Fig. 4C). Together with growth assays, these data suggest that in yeast cells, TAK1 functions as a Snf1-activating kinase and does so largely independently of TAB1. We cannot exclude the possibility that a native yeast protein functionally substitutes for TAB1, but no yeast sequence homolog is evident. TAK1 Catalytic Activity Is Required for Activation of Snf1 Protein Kinase—To determine whether the effects of TAK1 in yeast cells were due to the catalytic activity of TAK1, we introduced a mutation altering Lys-63 to Trp, which was previously shown to abolish catalytic activity of TAK1 (23Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1187) Google Scholar). The kinase-dead mutant p
ASK1
MAP2K7
Cyclin-dependent kinase 9
c-Raf
Cyclin-dependent kinase 4
MAPK14
Cite
Citations (457)
Journal Article Regulation of a Mitogen-Activated Protein Kinase Kinase Kinase, MLTK by PKN Get access Mikiko Takahashi, Mikiko Takahashi Search for other works by this author on: Oxford Academic PubMed Google Scholar Yusuke Gotoh, Yusuke Gotoh Search for other works by this author on: Oxford Academic PubMed Google Scholar Takayuki Isagawa, Takayuki Isagawa Search for other works by this author on: Oxford Academic PubMed Google Scholar Tamako Nishimura, Tamako Nishimura Search for other works by this author on: Oxford Academic PubMed Google Scholar Emiko Goyama, Emiko Goyama Search for other works by this author on: Oxford Academic PubMed Google Scholar Hon-Song Kim, Hon-Song Kim Search for other works by this author on: Oxford Academic PubMed Google Scholar Hideyuki Mukai, Hideyuki Mukai Search for other works by this author on: Oxford Academic PubMed Google Scholar Yoshitaka Ono Yoshitaka Ono Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 133, Issue 2, 1 February 2003, Pages 181–187, https://doi.org/10.1093/jb/mvg022 Published: 01 February 2003
MAP2K7
c-Raf
ASK1
Cyclin-dependent kinase 9
Cyclin-dependent kinase 4
Cite
Citations (33)