WO2008005418A2 - Assays for tak1 activity - Google Patents

Abstract

This invention discloses methods to identify modulators of TAK1 activity comprising measuring TAK1 activity or measuring TAK1- dependent Snf1/AMPK activity. Further disclosed are methods to measure TAK1 activity comprising measuring phosphorylation of an AMPK substrate or an increase in AMPK activity resulting from an increase in TAK1 activity. Methods to treat obesity, diabetes and diseases characterized by inflammation by administering compounds that modulate TAK1 are described also.

Description

ASSAYS FOR TAKl ACTIVITY

This invention was made with support under . United States

Government Grant NO.DK070653 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application, various publications are referenced by first author's last name and year of publication in parenthesis. Full citations for these publications may be found listed alphabetically at the end of the description section and preceding the claims section. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

BACKGROUND OF INVENTION

TGF-β-activated protein kinase (TAKl) is a member of the MAPK kinase kinase superfamily (MAPKKK7) and is widely conserved in eukaryotes (Adhikari et al., 2007, Kyriakis et al., 2001). TAKl is found in most tissues and cell types and is regulated in response to a variety of signals and stresses; it is essential for viability in mice (Shim et al, 2005) . TAKl was first identified in

1995 as a mediator of TGF-β signaling in mammalian cells (Yamaguchi et al . , 1995) and was soon shown to participate in bone morphogenetic protein signaling in Xenopus development

(Shibuya et al., 1998) and in Wnt signaling (Ishitani et al . ,

2003). Mammalian TAKl is also activated by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) , interleukin-1 (IL-I), and IL-6, and by bacterial lipopolysaccharide (LPS). TAKl mediates the activation of transcription factors nuclear factor-KB

(NFKB) and activator protein-1 (AP-I) by activating IKB kinase β

(IKKβ) and the c-Jun N-terminal kinase (JNK) and stress-activated protein kinase p38 MAPK pathways (Lee et al., 2000, Ninomiya-Tsuji et al., 1999, Shirakabe et al., 1997, Takaesu et al., 2000, Takaesu et al., 2003, Wang et al., 2001). TAKl directly phosphorylates IKKβ and the MAPK kinases MKK4, MKK3, and MKK6 (Moriguchi et al., 1996, Ninomiya-Tsuj i et al., 1999, Sakuri et al., 1999, Shirakabe et al., 1997, Wang et al., 2001, Yamaguchi et al . , 1995). Its role in stress responses is conserved in Drosophila, where the ortholog is an LPS- and hyperosmotic stress- responsive MAPK kinase kinase required for innate immunity (Chen et al., 2002, Vidal et al . , 2001).

TAKl has complex roles in cellular signaling and regulation, as it responds to a variety of upstream signals and influences a variety of downstream processes, when activated by a particular signal, TAKl selects specific downstream effector kinases via interaction with other proteins. For example, the protein TA02 directs TAKl to JNK rather than IKK in response to osmotic stress (Huangfu et al., 2006) . Similarly, studies of the IL-I response indicate a structured signaling pathway in which the TAKl-binding protein TAB2 , or the closely related protein TAB3, functions as an adaptor that links TAKl to TNF-α receptor-associated factor (TRAF) family member TRAF6 by binding to polyubiquitin chains generated in response to IL-I (Cheung et al., 2004, Ishitani et al., 2003, Kanayama et al . , 2004, Takaesu et al . , 2000, Wang et al . , 2001). Another TAKl-binding protein, TABl, increases TAKl autophosphorylation and catalytic activity and is essential for activation of TAKl in some contexts, for example, when TAKl is overexpressed (Kishimoto et al, 2000, Shibuya et al., 1996). TABl, TAB2 , and TAB3 are phosphorylated by p38α, which mediates negative regulation of TAKl in a feedback loop (Cheung et al., 2003, Cheung et al, 2004) .

TAKl is clearly central to signaling and regulation in normal cells and -also during development. Moreover, because it mediates cytokine and stress signaling through the NF-KB and JNK/p38 MAPK pathways, which in turn upregulate the expression of proinflammatory genes, TAKl is important in many human chronic inflammatory diseases, such as rheumatoid arthritis. It is also important in cardiovascular disease, as both the NF-KB and JNK/p38 MAPK signaling pathways have roles in atherosclerosis (Li et al, 2005). Moreover, inflammation is associated with obesity and type 2 diabetes; obesity is accompanied by the accumulation of macrophages in adipose tissue, where macrophage-related inflammatory pathways contribute to obesity-induced insulin resistance (Weisberg et al . , 2003, Xu et al., 2003). Thus, understanding the regulation and function of this kinase is relevant to the pathogenesis of many different diseases.

SUMMARY OF INVENTION

Disclosed within are methods to identify modulators of transforming growth factor-β-activated protein (TAKl) activity- comprising measuring TAKl activity or measuring TAKl-dependent Snfl/AMPK activity. Further disclosed are methods to measure TAKl activity comprising measuring phosphorylation of an AMPK substrate or an increase in AMPK activity resulting from an increase in TAKl activity. Methods to treat obesity, diabetes and diseases characterized by inflammation by administering compounds that modulate TAKl are disclosed also.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. depicts the strategy used for the identification of . mammalian Snf-1-activating kinases in yeast.

Figure 2. depicts the growth of mutant yeast cells expressing TAKl. Cultures expressing the indicated proteins were spotted with 5-fold serial dilutions on selective synthetic complete (SC) medium containing 2% glucose,

2% raffinose and the respiratory inhibitor antimycin A (lμg/mL) , or 2% glycerol plus 3% ethanol. Plates were incubated at 30⁰C and were photographed after 3 days or for raffinose plates, after 6 days. Panel A depicts snflόϊ.10 cells expressing GAD-Snfl, GAD-TAKl, or GAD

(vector) . Panels B and C depict saklΔ. tos3Δ elmlΔ cells expressing HA-CaMKKo-, HA-TAKl, HA-TAK1K63W, HA-Sakl, or HA (vector) and either LexA-TABl or LexA (vector) . Additional transformants expressing TAKl and TABl and transformants expressing TAK1K63W from three independent mutant plasmids were tested on raffinose with similar results .

Figure 3. depicts the assays used for Snfl catalytic activity. HA- CaMKK*, HA-TAKl, HA-TAK1K63W, LexA-TABl , or HA (vector) were expressed in saklAtos3AelmlA cells. Panels A and B depict phosphorylation of the SAMS peptide by partially purified Snfl. Extracts were prepared from two independent transformants, and each extract was assayed twice, with dilutions. Values are averages of four assays. For TAK1K63W, three transformants carrying independent mutant plasmids were used. In panels C and D, fractions assayed above were subjected to immunoblot analysis with anti-Snfl, anti-HA, and anti-LexA antibodies. Lane numbers correspond to assays numbered in panels A and B. Figure 4. depicts the phosphorylation of recombinant Snfl catalytic domain. In panels A through C, saklΔtos3Δ elmlΔ cells expressed HA-TAKl (+) , LexA-TABl (+) , HA {-) , or^' LexA

(-) , as indicated. Cell extracts were prepared, and proteins were immunoprecipitated with anti-HA antibody and incubated with bacterially expressed GST-SnflKD-

K84R, GST-SnflKD-T210A, or no substrate {None) and [J-

³²P]ATP. In panel A, products were separated by SDS-PAGE and detected by autoradiography. The lower panel shows a longer exposure. Molecular size markers (in kDa) are indicated. Arrowheads indicate GST-SnflKD substrate; asterisks indicate HA-TAKl. Panel B depicts the Coomassie Blue staining of the gel shown in panel A. In panel C, samples of the immunoprecipitated proteins used in panel A were immunoblotted with antiphospho-

Thr-172-AMPK antibody to detect phosphorylated Thr-210 (pT210) of Snfl and with anti-HA. In panel D, recombinant His-tagged TAKl-TABl fusion protein was incubated with substrates, as in panel A, and with cold ATP. Mock incubations with no added TAKl-TABl (-) were carried out as controls for specificity of the antibody. Proteins were immunoblotted with anti- phospho-Thr-172-AMPK and anti-His antibodies; Snfl has a stretch of His residues.

Figure 5. depicts the effects of TAKl in elmlΔ and saklΔ yeast cells. In panel A, θlmlΔ cells expressing HA-TAKl, HA- TAK1K63W, or HA (vector) and LexA-TABl or LexA (vector) were grown on selective SC+2% glucose and were imaged by differential interference contrast (DIC) . In panel

B, saklΔ cells expressing Gal83-GFP, HA-TAKl, and LexA- TABl were grown in selective SC+2% glucose and shifted to 0.05% glucose for 10 min. Nuclei were stained with 4 ' , 6-diamidino-2-phenylindole [DAPI). GFP fluorescence, 4 ' , 6-diamidino-2-phenylindole staining, and differential interference contrast are shown. Cells were viewed using a Nikon Eclipse E800 fluorescence microscope, and images were taken with an OrcalOO (Hamamatsu) camera by using Open Lab (Improvision) software.

Figure 6. depicts the phosphorylation of the recombinant AMPK catalytic domain. In panels A and B, saklΔtos3ΔelmlΔ cells expressed HA-TAKl (+) , LexA-TABl (+) , HA (-) , or LexA {-) , as indicated. Proteins were immunoprecipitated from cell extracts with anti-HA and were incubated with bacterially expressed AMPK-KD-WT, AMPK-KD-T172A, or no substrate {None) and [T-³²P]ATP. In panel A, products were separated by SDS-PAGE and detected by autoradiography. Molecular size markers (in kDa) are indicated. An arrowhead indicates substrate; an asterisk indicates HA-TAKl. In panel B, samples of the immunoprecipitates used in panel A were immunoblotted with anti-HA to detect HA-TAKl. In panel C, recombinant TAKl-TABl fusion protein was incubated with substrates, as in panel A, and with cold ATP.

Proteins were immunoblotted with anti-phospho-Thr-172- AMPK to detect phosphorylated Thr-172 (pT172) . The blot was reprobed with anti-AMPKα.

Figure 7 depicts the phosphorylation of Thr-172 of AMPK in HeIa cells. In panel A and B, HeLa cells were transfected with plasmids to transiently express TAKl, TAK1K63W, or TABl or with vector pCMV-FLAG2. At the indicated times after transfection, cells were transferred to serum- free medium for 4 h (3O h after transfection) or 14 h (12, 18, and 24 h after transfection) . In panel A, cell lysates (15 μg) were subjected to immunoblot analysis with anti-phospho-Thr-172 {pTl72) -specific antibody and ECL Advance. Blots were reprobed with anti-AMPKα, anti- TAKl, and anti-FLAG. In panel B, transfected HeLa cells were transferred to serum-free medium for 4 h and treated with 0.5 M sorbitol or 1 ΠM hydrogen peroxide for 15 min before lysis. Cell lysates were analyzed as in panel A. In panel C, HeLa cells were subjected to serum-free medium for 14 h prior to treatment with TNF- α or IL-lβ (10 ng/ml) for 2, 5, or 10 min. Control cells were untreated (0 min) . Cell lysates were analyzed as above.

Figure 8^_ depicts the radicicol inhibition of TAKl in yeast cells. Following transformation of sa.klΔtos3ΔelmlΔ yeast cells with a TAKl encoding plasmid, the cells were grown in SC plus 2% glucose and then shifted to SC plus 2% raffinose plus lμg/mL antimycin A with the indicated concentrations of radicicol. After 72 hours, the OD_60O was measured. Averages are for two transformants .

Figure 9. shows that radicicol has no effect on yeast growth in glucose. Following transformation of sa.klΔtos3ΔelmlΔ yeast cells with a TAKl encoding plasmid, the cells were grown in SC plus 2% glucose with indicated concentrations of radicicol. After 72 hours, the OD₆oo was measured. Averages are for two transformants .

Figure 10. shows that radicicol does not inhibit the Sakl protein kinase. Following transformation of saklΔtos3ΔelmlΔ yeast cells with a Sakl encoding plasmid, the cells were grown in SC plus 2% glucose and then shifted to SC plus 2% raffinose plus lμg/mL antimycin A with the indicated concentrations of radicicol. After 72 hours,, the OD₆₀₀ was measured. Averages are for two transformants .

Figure 11. shows the radicicol-mediated reversal of the normalization of yeast cell shape conferred by TAKl expression. Following transformation of saklΔtos3ΔelmlΔ yeast cells with a TAKl encoding plasmid, the cells were grown in SC plus 2% glucose with indicated concentrations of radicicol. The results of this experiment are shown photographically. The left panel shows yeast cells treated with 25μM radicicol and the right panel shows yeast cells treated with 25nM radicicol.

Figure 12. depicts the TABl-mediated improvement in TAKl function in yeast cells. Yeast saklΔtos3ΔelmlΔ cells were transformed with the indicated plasmids and spotted on glucose plates, raffinose plates or raffinose plus lμg/mL antimycin plates (raffinose + AntA) . "TAKl" expresses TAKl from a high copy number plasmid. 'TAKl.cen" expresses TAKl from a low copy number plasmid. The results of this experiment are shown photographically.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed is a method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising contacting a cell with the compound being tested as a modulator of TAKl activity, measuring TAKl-dependent Snfl/AMPK activity of the cell and comparing the TAKl-dependent Snfl/AMPK activity of the cell contacted with the compound being tested to the TAKl-dependent Snfl/AMPK activity of a control cell, wherein a difference between the TAKl-dependent Snfl/AMPK activity of the cell contacted with the compound being tested and the TAKl- dependent Snfl/AMPK activity of the control cell identifies the compound being tested as a modulator of TAKl activity.

In one embodiment, the cell, or the control cell or each is a yeast cell. In a further embodiment, the yeast cell is a saklΔtos3AβlmlA mutant. In a still further embodiment, the yeast cell overexpresses TAKl. In another embodiment, the yeast cell expresses low levels of TAKl. In a further embodiment, the yeast cell also expresses a known TAKl activator. In a further embodiment, the known TAKl activator is TABl, TAB2 , TAB3, both TABl and TAB2, or both TABl and TAB3. In another embodiment, the control cell is contacted with an inhibitor of TAKl. In a further embodiment, the inhibitor is radicicol. In one embodiment, the TAKl-dependent Snfl/AMPK activity is growth of the yeast cell in medium requiring Snfl/AMPK activity, and wherein a decrease in growth of the yeast cell contacted with the compound being tested in medium requiring Snfl/APMK activity as compared to the TAKl- dependent Snfl/AMPK activity of the control cell identifies the compound being tested as an inhibitor of TAKl. In another embodiment, the TAKl-dependent Snfl/AMPK activity is growth of the yeast cell in medium requiring Snfl/AMPK activity, and wherein an increase in. growth of the yeast cell contacted with the compound being tested in medium requiring Snfl/APMK activity as compared to the TAKl-dependent Snfl/AMPK activity of the control cell identifies the compound being tested as an activator of TAKl. In either embodiment, the medium requiring Snfl/AMPK activity comprises raffinose.

In another embodiment, the cell, or the control cell, or each is a mammalian cell. In a further embodiment, the mammalian cell is a HeLa cell. In another embodiment, the cell expresses low levels of TAKl. In a further embodiment, the cell also expresses a known TAKl activator. In a still further embodiment, the known TAKl activator is TABl, TAB2 , TAB3, both TABl and TAB2 , or both TABl and TAB3. In another embodiment, the control cell is contacted with an inhibitor of TAKl. In a further embodiment, the inhibitor is radicicol. In one embodiment, the TAKl dependent Snf-1/AMPK activity is AMPK phosphorylation in the cell, and wherein a decrease in the level of AMPK phosphorylation in the cell contacted with the compound being tested as compared to the level of AMPK phosphorylation in the control cell identifies the compound as an inhibitor of TAKl. In a different embodiment, the control cell is contacted with an activator of TAKl. In a further embodiment, the activator is a cytokine. In another embodiment, the activator is lipopolysaccharide . In a further embodiment, the TAKl dependent Snf-1/AMPK activity is AMPK phosphorylation in the cell , and wherein an increase in the level of AMPK phosphorylation in the cell contacted with the compound being tested as compared to the level of AMPK phosphorylation in the control cell identifies the compound as an activator of TAKl.

Also disclosed is a method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) comprising contacting a group of cells in suspension with the compound being tested as a modulator of TAKl activity, measuring the amount of cells settling out suspension from the group of cells and comparing the amount of cells settling out of suspension from the group of cells contacted, with the compound being tested to the amount of cells settling out of suspension from a control group of cells or measuring the amount of cells staying in suspension from the group of cells and comparing the amount of cells staying in suspension from the group of cells contacted with the compound being tested to the amount of cells staying in suspension from a control group of cells, wherein a difference in the amount of cells settling out of suspension from the group of cells contacted with the compound being tested as compared to the amount of cells settling out of suspension from the control group of cells or a difference in the amount of cells staying in suspension from the group of cells contacted with the compound being tested as compared to the amount of cells staying in suspension from the control group of cells identifies the compound as a modulator of TAKl .

In one embodiment, the group of cells, or the control group of cells, ■ or each group is a group of yeast cells. In a further embodiment, the yeast cells are elmlΔ or saklΔtos3AelmlA mutants.

In a further embodiment, the yeast cells overexpress TAKl. In another embodiment, the yeast cells express low levels of TAKl.

In a further embodiment, the yeast cells also express a known activator of TAKl. In a still further embodiment, the TAKl activator is TABl, TAB2 or " TAB3. In another embodiment, the control group of cells is contacted with a known TAKl inhibitor.

In a further embodiment, the TAKl inhibitor is radicicol.

In one embodiment, an increase in the amount of cells settling out of suspension from the group of cells contacted with the compound being tested as compared to the amount of cells settling out of suspension from the control group of cells identifies the compound being tested as an inhibitor of TAKl. in another embodiment, a decrease in the amount of cells settling out of suspension in the cells contacted with the compound being tested is greater than the amount of cells settling out of suspension in the control cells identifies the compound being tested as an activator of TAKl.

Further disclosed is a method for detecting transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising providing a sample comprising TAKl, contacting said sample with a TAKl substrate kinase under conditions permitting phosphorylation of the substrate by TAKl and determining if a phosphate is incorporated into the TAKl substrate kinase wherein the incorporation of phosphate into the TAKl substrate kinase indicates TAKl activity. In one embodiment, the substrate kinase is or is derived from AMPK. In a further embodiment, the AMPK comprises heterotrimeric AMPK. In a still further embodiment, the AMPK comprises mouse AMPK, rat AMPK or human AMPK.

In one embodiment, incorporation of the phosphate into AMPK is determined by quantitating the activity of the AMPK. In a further embodiment, AMPK activity is quantified by incorporation of a phosphate into a SAMS peptide.

In one embodiment, incorporation of phosphate into AMPK is determined by phosphorylation of T-172 of AMPK. In a further embodiment, comprising quantifying the amount of phosphate incorporated per mol of AMPK, wherein the phosphate level in AMPK indicates the level of TAKl present in the sample.

Yet further disclosed is a method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising providing a first sample comprising a TAKl and second sample comprising a TAKl, contacting the first sample with the compound to be tested as a modulator of TAKl activity, contacting each of the first and second samples with a substrate of TAKl under conditions permitting phosphorylation of the substrate, quantitating the incorporation of a phosphate into the substrate in the first and second samples and comparing the incorporation of phosphate into the substrate in the first sample with the incorporation of phosphate into the second sample, wherein a difference in the incorporation of phosphate into the substrate between the first and second samples identifies the compound as a modulator of TAKl activity. In one embodiment, substrate is a substrate kinase. in a further embodiment, the substrate kinase is or is derived from AMPK.

Also disclosed is a method of increasing AMPK activity comprising contacting AMPK with an agent which increases the activity of TAK in an amount effective to increase AMPK activity. In one embodiment, AMPK activity is increased by AMPK phosphorylation by TAKl. In a further embodiment, the contacting is performed in vitro. In another embodiment, the contacting is performed in vivo. In one embodiment, the agent which increases TAKl activity in the cell is TAKl, an agent causing overexpression of TAKl, causing overexpression TAKl activators, causing downregulation of TAKl inhibitors or causing a decrease in TAKl degradation.

Further disclosed is a method of treating obesity and diabetes in a subject comprising administering to the subject an agent which activates TAKl in said subject.

Yet further disclosed is a method of treating a disease characterized by inflammation in a subject comprising administering to the subject an agent which inhibits TAKl in the subject. In one embodiment, the disease characterized by inflammation includes arthritis, systemic lupus erythematosus, psoriasis, asthma, inflammatory bowel disease and hypercardiomyotrophy.

Also disclosed is a use of an agent that increases TAKl activity for the manufacture of a medicament for treating obesity or diabetes in a subject.

Further disclosed- is a use of an agent that inhibits TAKl activity for the manufacture of a medicament for treating a disease characterized by inflammation in a subject. In one embodiment, the disease characterized by inflammation includes arthritis, systemic lupus erythematosus, psoriasis, asthma, inflammatory bowel disease and hypercardiomyotrophy. Terms

Transforming growth factor-β activated kinase (TAKl) is a member of the mitogen activated protein (MAP) kinase kinase kinase family. This enzyme phosphorylates and activates its downstream protein kinase.

AMP-activated protein kinase (AMPK) is a protein kinase involved in regulating glucose and lipid metabolism, maintenance of cellular energy homeostasis and cellular stress responses.

As used herein, "Snfl" shall mean the "Snfl protein kinase" which is a heterotrimeric yeast protein comprising a catalytic subunit (Snfl/α) , and two regulatory subunits (β and Snf4/γ) . The Snfl protein kinase is the yeast ortholog of AMPK.

"TABl", "TAB2" and "TAB3" are TAKl- binding proteins 1, 2 and 3, respectively. These proteins complex with TAKl to activate TAKl.

A "substrate kinase" shall have its ordinary meaning. In particular, the 'kinase' means a recognizable kinase by sequence comparison, such as possessing kinase signature motif (s) as defined by Hunter (see, eg. Hanks, Quinn and Hunter Science vol 241 p.42) . 'Kinase' does not imply exclusion of kinase dead variants or catalytically inactive fragment (s), but is used to describe that family of proteins which are ordinarily classified as kinases with reference to Hanks et al. Kinase means protein and/or sugar and/or lipid kinase, preferably protein kinase.

The phrase "conditions that permit phosphorylation" refers to those conditions, for example, of buffer or pH, ionic concentrations, temperature, co-factor availability, and ATP or other phosphate donor concentration, under which phosphorylation of a given substrate will occur if a kinase enzyme is present in active form. As used herein "increasing TAKl activity" shall mean increasing the concentration by providing more TAKl protein, inducing TAKl activity by, for example, overexpressing TAKl or overexpressing TAKl activators to increase TAKl autophosphorylation, including but not limited to TABl, TAB2 and/or TAB3. Other known activators of TAKl include the receptor activator of NF-KB ligand (RANKL)

(Mizukami et al, 2002) , receptor interacting protein 2

(RIP2) (Windheim et al . , 2007) and tumor necrosis factor receptor associated factor 6 (TRAF6) (Mizukami et al, 2002) . Increasing the activity of TAKl can also occur by decreasing degradation of TAKl or reducing the phosphorylation of TABl, TAB2 and TAB3 by p38α, which mediates the negative regulation of TAKl (references 9 and 10 from grant) . TAKl activity can also be increased by treating cells with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) , interleukin-1 (IL-I) and IL-6. Treating cells with bacterial lipopolysaccharide (LPS) can increase TAKl activity also.

As used herein, "compounds that modulates TAKl activity" shall mean a compound that either directly or indirectly modulates the enzymatic activity of TAKl. Examples of such compounds may include, but are not limited to proteins that activate TAKl including TABl, TAB2 or TAB3. Modulators of TAKl may also include compounds that decrease TAKl activity which may include p38α, which inhibits TAKl activity by phosphorylating the TAB proteins to prevent their interaction with TAKl. Compounds may also include small molecule or chemical inhibitors. For example, the chemical compound 5Z-7-oxozeaenol is a specific TAKl inhibitor (Ninomiya-Tsuj i J, et al, 2003). When added to mammalian cells, 5Z-7-oxozeaenol efficiently prevented activation of TAKl targets, including p38, JNK and NF-KB. In vivo, 5Z-7-oxozeaenol reduced inflammation, indicating that TAKl is a necessary element of the inflammatory pathway. In addition, radicicol is a known small molecule inhibitor of TAKl (Ninomiya-Tsuji J, et al, 2003) , although it also inhibits heat shock protein 90 (Hsp90) (Sharma, SV., et al. 1998), a chaperone in mammalian cells, so it is not specific for TAKl. Compounds may also include dominant negative forms of the TAKl kinase including the kinase negative TAKl . Compounds may also include small interfering or small hairpin RNA molecules (siKNA and shRNA, respectively) or antibodies directed against TAKl.

As used herein, ^wTAKl-dependent Snfl/AMPK activity" shall mean modification of Snfl or AMPK or a homolog thereof resulting from the incorporation of a phosphate into Snfl or AMPK or a homolog thereof by TAKl. The incorporation of a phosphate can be measured by indirect methods, for example, a change in cell phenotype such as growth in medium requiring Snfl/AMPK activity. Medium which requires Snfl/AMPK activity may include any medium comprising any carbon source requiring Snfl/AMPK activity for utilization including but not limited to raffinose, raffinose plus antinmycin A. Further, the concentration of the carbon source can be titrated as well. The incorporation of a phosphate into Snfl or a homolog of Snfl such as AMPK can be measured directly by in vitro kinase assays or Western blotting.

As used herein, "overexpress" shall mean that the amount of a protein is expressed at a high level in a cell. The method of expressing high levels of protein shall be known to those skilled in the art; however as defined here within, overexpression of the protein can be achieved by transforming or transfecting the cell with a high copy number plasmid encoding the protein of interest. Western blotting using either a polyclonal or monoclonal antibody directed against the protein of interest can be used to measure the amount of protein in the cell.

As used herein, "high copy number plasmid" shall mean that the plasmid contains an origin of replication which maintains the plasmid under relaxed conditions allowing for increased number of copies of the plasmid consequently increasing the level of protein expression (see, eg, Sambrook, J. et al., eds . Molecular Cloning: A Handbook. Cold Spring Harbor Laboratory Press, 1989) .

As used herein, "low levels" shall mean that the amount of protein expressed in a cell is expressed off a low copy number plasmid. The methods used to express low levels of protein shall be known to those skilled in the art; however as defined here ^v within, low levels of protein expression can be achieved by transforming or transfecting the cell with a low copy number plasmid encoding the protein of interest. Western blotting using either a polyclonal or monoclonal antibody directed against the protein of interest can be used to measure the amount of protein in the cell .

As used herein, a "low copy number plasmid" shall mean that the plasmid contains an origin of replication which maintains the plasmid under stringent conditions thereby restricting the number of copies of the plasmid consequently limiting the level of protein expression! see, eg, Sambrook, J. et al., eds. Molecular Cloning: A Handbook. Cold Spring Harbor Laboratory Press, 1989) .

As used herein, "TAKl. cen" refers to a low copy number plasmid used to express low levels of TAKl in a cell.

"Treating" a disorder shall mean slowing, stopping or reversing the disorder's progression.

"Compound" shall mean any chemical entity, including, without limitation, a glycomer, a polypeptide, a fusion protein, a peptidomimetic , a carbohydrate, a lipid, an antibody, a lectin, a nucleic acid, a small molecule, and any combination thereof.

"Subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate such as a human. "Administering" a compound can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, nasally, via the cerebrospinal fluid, via implant, transmucosally, transdermalIy, intramuscularly, intraocularIy, topically and subcutaneousIy. The following delivery systems, which employ a number of routinely used pharmaceutically acceptable carriers, are only representative of the many embodiments envisioned for administering compositions according to the instant methods .

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering compounds (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA' s) . Implantable systems include rods and discs , and can contain excipients such as PLGA and polycaprylactone .

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating compounds (e.g., starch polymers and cellulosic materials) and lubricating compounds (e.g., stearates and talc) .

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) . Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions , liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such, as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending compounds (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking compounds, coating compounds, and chelating compounds (e.g., EDTA).

In the practice of the method, administration may comprise daily, weekly, monthly or hourly administration, the precise frequency being subject to various variables such as age and condition of the subject, amount to be administered, half-life of the compound in the subject, area of the subject to which administration is desired and the like.

^■"Therapeutically effective amount" of a compound means an amount of the compound sufficient to treat a subject afflicted with a disorder or a complication associated with a disorder. The therapeutically effective amount will vary with the subject being treated, the condition to be treated, the compound delivered and the route of delivery. A person of ordinary skill in the art can perform routine titration experiments to determine such an amount. Depending upon the compound delivered, the therapeutically effective amount of compound can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions) . Desired time intervals of multiple amounts of a particular compound can be determined without undue experimentation by one skilled in the art.

"Pharmaceutically acceptable carriers" are well known to those skilled in the art and include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may include, but are not limited to, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol', polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For administration in an aerosol, such as for pulmonary and/or intranasal delivery, an agent or composition is preferably formulated with a nontoxic surfactant, for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery. Preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like may also be included with all the above carriers .

The compound may be conjugated to a carrier. The peptide or compound may be linked to an antibody, such as a Fab or a Fc fragment for specifically targeted delivery. The carrier may be a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.

When administered, compounds are often cleared rapidly from the circulation and may therefore elicit relatively short-lived pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive compounds may by required to sustain therapeutic efficacy. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al . , 1981; Newmark et al . , 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound adducts less frequently or in lower doses than with the unmodified compound.

"Peptide," "polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may comprise either partial or full-length sequences of amino acid residues .

"Amino acid residue" means an individual monomer unit of a polypeptide chain, which result from at least two amino acids combining to form a peptide bond.

"Amino acid" means an organic acid that contains both an amine group and a carboxyl group.

The abbreviations used herein for amino acids are those abbreviations which are conventionally used: A=Ala=Alanine; R=Arg=Arginine; N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine; E=Glu=Gutamic acid; G=Glγ=Glγcine; H=His=Histidine; I=Ile=Isoleucine; L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine,- F=Phe=Phenyalanine; P=Pro=Proline; Ξ=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan; Y=Tyr=Tyrosine; V=,VaI=Valine. The amino acids may be L- or D- amino acids. An amino acid may be replaced by a synthetic amino acid which is altered so as to increase the half-life of the peptide or to increase the potency of the peptide, or to increase the bioavailability of the peptide.

EXPERIMENTAL DETAILS

Example 1: TAKl activates Snfl and AMPK

Genetic Selection for Mammalian Snfl-activating Kinases in Yeast— The saklAtos3AelmlΔ mutant yeast strain lacks all three native Snfl 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, mammalian cDNAs that allow saklAtos3AeImIA cells to grow on raffinose were selected, as shown schematically in Figure 1. A library of mouse 17-day embryo cDNAs fused to the Gal4 activation domain (GAD) in a yeast expression vector were used to transform saklAtos3AelmlA yeast cells and then selected for growth on raffinose. The cDNA plasmid was recovered from Snf⁺ colonies by passage through Escherichia coli, retransformed saklAtos3AelmlA yeast cells to confirm that the cDNA conferred a Snf⁺ phenotype, and identified by sequencing. In a screen of 5 X 10⁶ transformants, 49 cDNA clones expressing LKBl, five expressing CaMKKβ, and six expressing TAKl, also known as MAPKKK7 (Yamaguchi et al., 1995) were recovered. This selection also yielded seven cDNAs expressing transcription factors, which were not characterized further, but none expressing CaMKKa, which was previously showed to function in yeast (Hong et al., 2005; see Figure 2B). The recovery of LKBl and CaMKKβ, which, are both, known AMPK kinases, validates this approach. TAKl is thus a candidate for a Snf1-activating kinase and potentially an AMPK kinase.

Growth Phenotype Conferred by TAKl Requires Snfl Protein Kinase—To confirm that the ability of TAKl to confer growth on raffinose requires Snfl protein kinase, a cDNA plasmid expressing GAD-TAKl was used to transform snfIA mutant cells. The transformants did not grow on raffinose (Figure 2A) , indicating that TAKl requires Snfl protein kinase to confer a Snf⁺ phenotype and does not function by bypassing Snfl. In control experiments, expression of Snfl in the mutant cells restored growth.

The cDNA clones recovered from the library expressed TAKl with GAD fused to its N terminus. To exclude the possibility that this fusion protein had aberrant function, full-length TAKl, tagged with a triple-HA epitope at its N terminus, was expressed from the yeast ADHl promoter of vector pWS93. Expression of this HA-tagged TAKl allowed saklAtos3AeImIA cells to grow on raffinose (Figure 2B) and on glycerol plus ethanol (Figure 2C) ; HA-TAKl was used in all subsequent experiments. TABl, a TAKl-binding protein identified in the two-hybrid system, increases the catalytic activity of TAKl (Shibuya et al . , 1996); however, TAKl acts independently of TABl in some signaling pathways in mammalian cells (Shim et al., 2005) . Coexpression of LexA-TABl from the ADHl promoter did not improve growth of saklAtos3A.elmlA cells on raffinose (Figure 2B) , although some improvement was noted on glycerol-ethanol (Figure 2C) ; expression was confirmed by immunoblot analysis (Figure 3C) . In addition, TAKl, with or without TABl, did not allow raffinose utilization by snfIA cells expressing mutant SnflT210A with the activation loop Thr-210 replaced by Ala, as predicted if TAKl functions by phosphorylating Thr-210.

TAKl Activates Snfl Protein Kinase in Vivo—To determine whether

TAKl activates Snfl protein kinase in vivo, Snfl catalytic activity was assayed in saklAtos3AelmlA mutant cells expressing HA- TAKl. 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 Snfl in wild-type cells. Cell extracts were prepared, and phosphorylation of a synthetic peptide substrate, the SAMS peptide, by partially purified Snfl protein kinase was determined. The presence of HA-TAKl in the mutant cells resulted in the activation of Snfl to levels similar to those caused by CaMKKa (Figure 3A) , which is roughly 2-fold reduced relative to wild type {Hong et al., 2005). Coexpression of LexA- TABl with HA-TAKl did not substantially increase activation of Snfl (Figure 3A) , consistent with the growth phenotypes (Figure 2) . Amounts of Snfl protein were similar in all assays, and coexpression of TABl did not result in elevated levels of TAKl, although TAKl appeared to stabilize TABl, as judged by immunoblot analysis (Figure 3C) . Together with growth assays, these data suggest that in yeast cells, TAKl functions as a Snf1-activating kinase and does so largely independently of TABl .

TAKl Catalytic Activity Is Required for Activation of Snfl Protein Kinase—To determine whether the effects of TAKl in yeast cells were due to the catalytic activity of TAKl, a mutation was introduced altering Lys-63 to Trp, which was previously shown to abolish catalytic activity of TAKl (Yamaguchi et al . , 1995). The kinase- dead mutant protein, TAK1K63W, was expressed (Figure 3D) but did not confer growth on raffinose, indicating that catalytic activity and not some other property of the protein is required (Figure 2C) . In accord with this result, TAK1K63W did not activate Snfl protein kinase activity in vivo, as judged by phosphorylation of the SAMS peptide (Figure 3B) . Thus, the function of TAKl as a Snfl- activating kinase in yeast requires its catalytic activity.

TAKl-TABl Phosphorylates the Activation Loop Thr-210 of Recombinant Snfl Catalytic Domain—-The ability of TAKl purified from yeast cells to phosphorylate the kinase domain of Snfl (SnfIKD) in vitro was assayed next. Two bacterially expressed, inactive forms of the Snfl catalytic domain, GST-Snf1KD-K84R, which has a substitution of the conserved Lys of the ATP-binding site, and GST- Snf1KD-T210A, which is mutant for the activation loop Thr-210 were used as substrates. HA-TAKl was expressed in saklΔtos3ΔelmlA cells in combination with LexA-TABl or LexA, immunoprecipitated with anti-HA antibody, and incubated with different substrates and [7- ³²P]ATP- TABl has been reported to stimulate the autophosphorylation of TAKl (Kishimoto et al . , 2000, Sakuri et al . ,

2000) . The presence of TABl increased the phosphorylation of SnflKD substrates (Figure 4A) but did not increase the recovery of TAKl

(Figure 4C) ; longer exposure revealed very weak phosphorylation of SnflKD by TAKl in the absence of TABl (Figure 4A, lower panel) .

These results stand in contrast to the minimal effect of TABl on activation of Snfl by TAKl in yeast cells; however, the substrate in vivo was full-length Snfl protein, presumably in the context of the heterotrimeric Snfl protein kinase complex.

Both SnflKD substrates were phosphorylated by TAKl with TABl, although SnflKD-T210A was phosphorylated less strongly than was SnflKD-K84R (Figure 4A) , and Coomassie Blue staining confirmed that both substrates were present at similar levels (Figure 4B) . These findings suggest that TAKl recognizes Thr-210, as well as other residues. To directly assess the phosphorylation of Thr-210, immunoblot analysis was carried out with anti-phospho-Thr-172- AMPK-specific antibody, which cross-reacts with phospho-Thr-210 of Snfl. This antibody detected SnflKD-K84R, but not SnflKD-T210A, indicating that TAKl phosphorylates Thr-210 in the activation loop (Figure 4C) .

To confirm that TAKl, and not a coprecipitating kinase, is responsible for phosphorylation of Thr-210, recombinant human TAKl- TABl fusion protein (TAKl residues 1-303 fused to TABl residues 437-end) (Sakuri et al., 2002) was incubated with the SnflKD substrates and cold ATP. Immunoblot analysis with phospho-Thr-172- AMPK antibody detected SnflKD-K84R but not SnflKD-T2l0A (Figure 4D) . These biochemical studies indicate that TAKl phosphorylates the activation loop Thr-210 of Snfl in vitro and, together with genetic evidence, suggest that TAKl functions directly as a Snfl- activating kinase in yeast cells in vivo. TAKl Restores Normal Cell Morphology in elmlA Cells—The Sn.fl protein kinase kinases Elml and Sakl have other roles in the cell besides activation of Snfl, therefore TAKl was tested for the ability to provide these distinct functions. The elml mutation is named for the elongated morphology of the mutant cells, which results from defects in cell cycle progression and has no apparent relationship to Snfl (Blacketer et al., 1993, Bouquin et al., 2000, Sreenivasan et al . , 2003, Sreenivasan et al., 1999). Expression of TAKl, with or without TABl, in elmlA cells restored normal cell morphology, whereas expression of TAK1K63W with TABl had no effect (Figure 5A) . Thus, TAKl functionally substitutes for Elml with respect to this phenotype. These findings suggest that TAKl and Elml phosphorylate substrate (s) that are not efficiently recognized by Sakl or Tos3. Neither LKBl nor CaMKK substituted for Elml in this regard {Hong et al . , 2005) .

The saklΔ mutation prevents the nuclear enrichment of Snfl protein kinase containing the Gal83 β subunit (Snfl-Gal83) in response to glucose limitation (Hedbacker et al . , 2004). Although activation of Snfl is required for this nuclear enrichment (Hedbacker et al., 2004), evidence suggests that Sakl also functions in another capacity, besides activating Snfl, to promote the nuclear enrichment of Snfl-Gal83 (Hong et al . , 2005). To test whether TAKl provides this function, TAKl and TABl were expressed in saklA. cells carrying a centromeric plasmid expressing Gal83-GFP from its native promoter. Cells were grown to mid-log phase in medium containing 2% glucose and shifted to 0.05% glucose for 10 min. Microscopic observation revealed no nuclear enrichment of Gal83-GFP (Figure 5B) . Together, these findings suggest that with respect to function, TAKl is more closely related to Elml than to Sakl.

Recombinant TAKl-TABl Fusion Protein Phosphorylates Thr-172 of AMPK Catalytic Domain—The above evidence that TAKl functions in vivo and ^■in vitro as a Snf1-activating kinase supports TAKl as a candidate for an AMPK kinase. To examine whether TAKl phosphorylates AMPK on the activation loop Thr-172 in vitro, the wild-type AMPK catalytic domain, AMPK-KD-WT, and a mutant version containing a replacement of Thr-172 with Ala, AMPK-KD-T172A were expressed in bacteria. The purified proteins were tested as substrates for Tos3, which phosphorylates AMPK on Thr-172 (Hong et al . , 2003). HA-Tos3 was expressed in saklΔtos3Δ.elmlA cells, immunoprecipitated with anti- HA, and incubated with the substrates and [7-³²P]ATP. AMPK-KD-WT, but not AMPK-KD-T172A, was phosphorylated.

Phosphorylation of AMPK-KD substrates by TAKl were assayed next. HA-TAKl was expressed in saklAtos3ΔelmlΔ cells in combination with LexA or LexA-TABl, immunoprecipitated, and incubated with both versions of AMPK-KD and [7-³²P]ATP. The presence of TABl increased the phosphorylation of both substrates (Figure 6A) , without increasing the recovery of TAKl (Figure 6B) . A mock immunoprecipitation with no cell extract gave results similar to the control sample with HA and LexA-TABl (Figure 6A) . TAKl, when coexpressed with TABl, phosphorylated AMPK-KD-WT more strongly than AMPK-KD-T172A, and close inspection revealed a doublet in the case of the wild-type substrate, suggesting that Thr-172 is one of the sites recognized.

To determine whether TAKl recognizes Thr-172, recombinant human TAKl-TABl fusion protein was incubated with AMPK-KD-WT and AMPK- KD-T172A and cold ATP. Immunoblot analysis with phospho-Thr-172- AMPK antibody detected AMPK-KD-WT but not the mutant protein lacking Thr-172 (Figure 6B) . Thus, TAKl phosphorylates the activation loop Thr-172 of AMPK in vitro.

Coexpression of TAKl and TABl in HeLa Cells Increases AMPK Phosphorylation—These findings suggest TAKl as a candidate for an AMPK kinase in mammalian cells. To explore this possibility, HeLa cells, which do not express the major AMPK kinase LKBl (Tiainen et al, 1999), were transfected with combinations of plasmids for transient expression of TAKl, TAK1K63W, and TABl from the vector pCMV-FLAG2. At 30 h after transfection, cells were subjected to serum-free medium for 4 h. Cell lysates, prepared by a rapid lysis procedure, were subjected to immunoblot analysis with anti-phospho- Thr-172-specific antibody, and blots were reprobed with anti-AMPKα, anti-TAKl, and anti-FLAG antibodies (Figure 7A) . Expression of TAKl alone had little or no effect, but coexpression of TAKl and TABl led to increased phosphorylation of Thr-172, whereas levels of AMPK catalytic subunit remained constant. Kinase-dead TAK1K63W, with TABl, did not increase phosphorylation of Thr-172. TAK1K63W was expressed at lower levels than TAKl, which was not the case in yeast (Figure 3D) , suggesting that in HeLa cells, the kinase-dead protein is either less stable or deleterious. Similar results were observed when cells were transferred to serum-free medium for 14 h at 12, 18, or 24 h after transfection (Figure 7A) . Coexpression of TAKl and TABl also increased phosphorylation of AMPK in HeLa cells treated with 0.5 M sorbitol or 1 iriM hydrogen peroxide for 15 min (Figure 7B) . Both treatments are known to activate AMPK, but hyperosmotic stress does not alter the AMP:ATP ratio (Fryer et al . , 2002) and has been shown to stimulate TAKl activity (Shirakabe et al, 1997, Cheung et al., 2003). Together, these findings indicate that coexpression of TAKl and TABl stimulates phosphorylation of AMPK in HeLa cells.

Several cytokines stimulate TAKl activity, including TGF-β, TNF-α, and IL-I (Yamaguchi et al . , 1995, Shirakabe et al., 1997, Ninomiya-Tsuj i et al., 1999, Cheung et al., 2003). Exposure to TGF- β induces phosphorylation of AMPK on Thr-172 in HeLa and HepG2 cells (Suzuki et al . , 2004, Suzuki et al. , 2005). To test the effects of TNF-α and IL-I, HeLa cells were subjected to serum-free medium for 14 h, treated with cytokine, and analyzed cell lysates by immunoblotting. In both cases, a modest increase in Thr-172 phosphorylation between 2 and 10 min (Figure 7C) was detected, consistent with the possibility that native TAKl phosphorylates AMPK.

Example 2 : Assay For Inhibitors of TAKl To test whether the known TAKl inhibitor radicicol can inhibit

TAKl in yeast cells, TAKl was overexpressed in the saklAtos3AelmlA strain. Growth of this yeast strain on alternative carbon sources

(raffinose or others) depends on the kinase activity of TAKl. Several concentrations of radicicol were tested to determine if growth inhibition would occur. Concentrations up to 2.5μM had no significant effect on cell growth. However, 25μM of radicicol did cause a significant reduction in the cell density, indicating that radicicol at the concentration of 25uM inhibited TAKl (Figure 8) .

To confirm that obtained results are consequences of a specific inhibition of TAKl by radiciol, rather than non-specific toxic effect of radicicol on yeast cells, two control experiments were performed. In the first experiment, yeast cells expressing TAKl were grown in glucose media. Under these conditions, cells do not require TAKl activity for growth. If radicicol has non-specific toxic effects, cells should not be able to grow in glucose media. It was found that there was no significant inhibition of growth up by 25μM of radicicol in glucose media (Figure 9) .

In a second control experiment, yeast cells expressed Sakl, a native yeast kinase necessary for growth in raffinose media. When Sakl expressing cells were exposed to radicicol in raffinose media, no growth inhibition occurred (Figure 10) . Both control experiments show that there is no significant toxic effect in yeast cells at the relevant concentrations of radicicol .

In an alternative assay for TAKl inhibitors, we can take advantage of the fact that expressing TAKl restores cell shape of the yeast saklΔ tos3Δ elmlΔ cells or elmlΔ cells. Restoration of the cell shape was prevented by radicicol at 25 microM (Figure 11) .

Example 3: An assay for inhibitors of TAKl/TABl interaction or inhibitors of TABl. In mammalian cells, overexpressing TAKl alone does not lead to the detectable activation of downstream targets. However, expressing TAKl together with TABl does increase kinase activity of TAKl and results in the activation of downstream pathways.

In order to reproduce similar behavior of TAKl and TABl in yeast cells, A TAKl plasmid that expresses TAKl at low levels (TAKl. cen) was constructed. When expressed alone from TAKl. cen plasmid, TAKl does not lead to the activation of the Snfl in saklAtos3AelmlΔ strain and therefore no growth occurs on alternative carbon sources. However, when TAKl (expressed from TAKl. cen) and TABl are co-expressed, growth of saklΔ tos3Δ elmlΔ strain on alternative carbon sources is restored (Figure 12) .

Materials and Methods

Yeast Strains—5. cerevisiae strains were W303-1A (MATa. ura3 txrpl ade2 his3 canl Ieu2) , MCY4908 (W303-1A snflAlO) , MCY5138 (MAOt* saklΔ.: :kanMX4 tos3&.~. ikanMXά elmlΔ.: :ADE2 ura3 trpl ade2 his3 canl Ieu2) , MCY5115 (MATα εaklA: : kanMX4 ura3 trpl ade2 his3 canl Ieu2) , and MCY5125 (W303-1A elmlΔ.: :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 (Gietz et al . , 2002) yeast strain MCY5138 (see Figure 1) . A total of 5 x 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 Snfl protein kinase; in control experiments, colonies expressing LKBl 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-TAKl, was recovered above. pRH104 (Hedbacker et al., 2004), pRHlO5 (Hong et al . , 2005), and pRH123 (Hong et al., 2005) express HA-Sakl, HA-Tos3, and HA-CaMKKa?, respectively, from vector pWS93 (Song et al., 1998). GAD-Snfl was expressed from pSGl (Jiang et al., 1996). pMM25 and pMM29 express HA-TAKl and LexA-TABl, respectively, from mouse cDNAs (Open Biosystems) cloned into pWS93 (Song et al., 1998) and pBTMllβ (Fields et al., 1989). 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 Crute et al, 1998), were transferred to vector pET32a (Novagen) to yield pMM45 and pMM57, respectively, expressing His-tagged proteins. pMM33, pMM35, and pMM37 express TAKl, TAK1K63W, and TABl, respectively, from vector pCMV-FLAG2 (Invitrogen) ; TAKl proteins were not recognized by anti-FLAG,^' although sequence analysis confirmed the FLAG tag. SnflT210A and Gal83, tagged with green fluorescent protein (GFP), were expressed from their native promoters on pKH43 (Hedbacker et al., 2004) and pRT13 (Hedbacker et al, 2004).

Analysis of Proteins—Proteins were separated by SDS-PAGE in 8% polyacrylamide. Immunoblot analysis was carried out with anti-Snfl

(Celenza et al . , 1986), monoclonal anti-HA (12CA5), anti-LexA

(Invitrogen) , anti-FLAG (Sigma) , anti-TAKl (Upstate) , anti-phospho- Thr-172-AMPK and anti-AMPK« (Cell Signaling Technologies) , and anti-Hisβ-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 Snfl 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, Snfl was partially purified, and phosphorylation of the synthetic peptide HMRSAMSGLHLVKRR (SEQ ID NO. 1) (SAMS peptide; Davies et al., 1989) was assayed as described (Woods et al., 1994, Hedbacker et al.,2004 ) . 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 (Davies et al., 1989).

Assay of Phosphorylation of Recombinant Snfl and AMPK Catalytic Domains—Glutathione S-transferase (GST) fusions to the mutant Snfl catalytic domains SnflKD-K84R and SnflKD-T210A were expressed in bacteria and purified as described (Hong et al., 2005). 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-TAKl and/or LexA-TABl 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 (Hong et al., 2003) . Kinases were assayed for phosphorylation of GST-SnflKD (3 μg) or AMPK-KD (0.5 μg) substrates using [T-³²P]ATP as described (Hong et al, 2005) . His-tagged recombinant human TAKl-TABl 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-rQ^{^}lutamine. Cells were transfected with DNAs (8 μg/6-cm dish) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. TNF-a and IL-lP were purchased from R&D Systems. Cells were lysed by the addition of ice-cold lysis buffer as described (Woods et al., 2005) , except without prior rinsing. Lysates were collected immediately and clarified by brief centrifugation in the cold.

DISCUSSION

TAKl has already been validated as a useful target for inflammatory disease; 5Z-7-oxozeaenol was identified as a selective inhibitor of TAKl catalytic activity and shown to relieve inflammation in a mouse model of picryl chloride-induced ear swelling (Ninomiya-Tsuji et al, 2003).

AMPK is a key energy sensor in mammalian cells and serves to maintain the cellular energy balance (Kahn et al . , 2005, Kemp et al . , 2003), and its S. cerevisiae ortholog is Snfl protein kinase (Hardie et al . , 1998, Mitchelhill et al., 1994, Woods et al., 1994) . Snfl protein kinase alters gene expression and metabolism in response to stress, particularly carbon stress, and is required for growth on nonpreferred carbon sources (Hardie et al., 1998). Activation of the Snfl/AMPK heterotrimeric enzymes requires phosphorylation of the conserved threonine in the activation loop of the catalytic subunit by an upstream kinase. There are three identified yeast upstream kinases, called Sakl , Tos3, and Elml

(Hong et al . , 2003, Nath et al . , 2003, Sutherland et al . , 2003), which led to the identification of LKBl as the first AMPK kinase (Hawley et al . , 2003, Hong et al . , 2003, Woods et al., 2003) and to later studies showing that Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) α and β are also AMPK kinases (Hawley et al, 2005, Hong et al., 2005, Hurley et al., 2005, Woods et al., 2005). Expression of LKBl and the CaMKKs in yeast, and biochemical analysis in vitro, revealed profound functional conservation between the yeast and mammalian Snfl/AMPK pathways (Hong et al . , 2005) .

Taking advantage of the conservation of the Snfl/AMPK pathway, the yeast system has been used to identify putative AMPK kinases by their function as Snf1-activating kinases. This genetic selection yielded two authentic AMPK kinases, LKBl and CaMKKβ, and a new candidate, TAKl. The utility of this genetic approach is that it is based on function. Although LKBl and CaMKK are homologous to the three yeast Snfl protein kinase kinases, TAKl was not identified as a candidate AMPK kinase on the basis of sequence similarity. In this study, a mouse embryo cDNA library was used, which may not represent the entire repertoire of AMPK kinases. Different libraries from other developmental stages or from specific tissues may yield additional AMPK kinases. Such kinases are potentially useful therapeutic targets in the AMPK pathway.

Genetic and biochemical evidence is presented that validates TAKl as a Snf1-activating kinase. This work demonstrates that recombinant TAKl-TABl phosphorylates AMPK on Thr-172 in vitro and that overexpression of TAKl and TABl stimulates phosphorylation of AMPK in HeLa cells. The stimulatory effects of TGF-β (Suzuki et al., 2004), TNF-or, and IL-I on phosphorylation of AMPK in HeLa cells are also in accord with the possibility that TAKl phosphorylates AMPK. Together, these findings support TAKl as a candidate for an authentic AMPK kinase in mammalian cells.

It should be noted that other work has connected TABl with AMPK. TABl interacts with the t*2 isoform of the catalytic subunit of AMPK in mouse heart, and activation of AMPK promoted the association of p38 MAPK with TABl in ischemic heart (Li et al . , 2005); however, TAKl was not implicated, and evidence suggests that TAKl and TABl have some independent roles (Shim et al., 2005).

Further, two assays for the identification of TAKl inhibitors and activators are described herein. Yeast saklΔtσs3AelπιlA cells expressing TAKl can be used to screen for modulators of TAKl activity as demonstrated through the use of the known TAKl inhibitor, radicicol. Treatment of these yeast cells with radiciol impaired the growth of the yeast in media wherein Snf-1 activity is required. There was no effect conferred by radiciol on the same strain of yeast grown in media containing glucose, which does not require Snfl activity for growth. Likewise, a second assay, which takes advantage of a phenotype wherein expression of TAKl in elmlΔ yeast restores normal yeast cell shape, can also be used to screen for modulators of TAKl activity. Again, treatment of these yeast cells with radicicol inhibited TAKl, resulting in a return to the misshapen yeast phenotype. These misshapen yeast also readily fall out of suspension in culture, another phenotype that can be used to screen for modulators of TAKl activity.

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Claims

What is claimed is :

1. A method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising:

(i) contacting a cell with the compound being tested as a modulator of TAKl activity; (ii) measuring TAKl-dependent Snfl/AMPK activity of the cell; and

(iii) comparing the TAKl-dependent Snfl/AMPK activity of the cell contacted with the compound being tested to the TAKl-dependent Snfl/AMPK activity of a control cell,

wherein a difference between the TAKl-dependent Snfl/AMPK activity of the cell contacted with the compound being tested and the TAKl-dependent Snfl/AMPK activity of the control cell identifies the compound being tested as a modulator of TAKl activity.

2. The method of claim 1, wherein the cell, or the control cell or each is a yeast cell.

3. The method of claim 2, wherein the yeast cell is a saklAtos3AelmlA mutant.

4. The method of any one of claims 2-3, wherein the yeast cell overexpresses TAKl .

5. The method of any one of claims 2-3, wherein the yeast cell expresses low levels of TAKl.

6. The method of any one of claim 2-5, wherein the yeast cell also expresses a known TAKl activator.

7. The method of claim 6 , wherein the known TAKl activator is

TABl, TAB2, TAB3, both TABl and TAB2 or both TABl and TAB3.

8. The method of any one of claims 1-7, wherein the control cell is contacted with an inhibitor of TAKl .

9. The method of claim 8, wherein the inhibitor is radicicol.

10. The method of any one of claims 2-9, wherein the TAKl- dependent Snfl/AMPK activity is growth of the yeast cell in medium requiring Snfl/AMPK activity, and wherein a decrease in growth of the yeast cell contacted with the compound being tested in medium requiring Snfl/APMK activity as compared to the TAKl-dependent Snfl/AMPK activity of the control cell identifies the compound being tested as an inhibitor of TAKl.

11. The method of any one of claims 2-9, wherein the TAKl- dependent Snfl/AMPK activity is growth of the yeast cell in medium requiring Snfl/AMPK activity, and wherein an increase in growth of the yeast cell contacted with the compound being tested in medium requiring Snfl/APMK activity as compared to the TAKl-dependent Snfl/AMPK activity of the control cell identifies the compound being tested as an activator of TAKl.

12. The method of any one of claims 10 or 11, wherein the medium requiring Snfl/AMPK activity comprises raffinose.

13. The method of claim 1, wherein the cell, or the control cell, or each is a mammalian cell.

14. The method of claim 13, wherein the mammalian cell is a HeLa cell.

15. The method of any one of claims 13 or 14, wherein the cell expresses low levels of TAKl .

16. The method of claim 15, wherein the cell also expresses a known TAKl activator.

17. The method of claim 16, wherein the known TAKl activator is TABl, TAB2 , TAB3, both TABl and TAB2 or both TABl and TAB3.

18. The method of any one claims 1 or 13-17, wherein the control cell is contacted with an inhibitor of TAKl.

19. The method of claim 18, wherein the inhibitor is radicicol.

20. The method of any one of claims 13-19, wherein the TAKl dependent Snfl/AMPK activity is AMPK phosphorylation in the cell, and wherein a decrease in the level of AMPK phosphorylation in the cell contacted with the compound being tested as compared to the level of AMPK phosphorylation in the control cell identifies the compound as an inhibitor of TAKl.

21. The method of any one of claims 1 or 13-17, wherein the control cell is contacted with an activator of TAKl.

22. The method of claim 21, wherein the activator is a cytokine.

23. The method of claim 21, wherein the activator is lipopolysaccharide.

24. The method of any one of claims 1, 13-19 or 21-23, wherein the TAKl dependent Snfl/AMPK activity is AMPK phosphorylation in the cell, and wherein an increase in the level of AMPK phosphorylation in the cell contacted with the compound being tested as compared to the level of AMPK phosphorylation in the control cell identifies the compound as an activator of TAKl.

25. A method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) comprising: (i) contacting a group of cells in suspension with the compound being tested as a modulator of TAKl activity; (ii) determining the amount of cells settling out suspension from the group of cells or the amount of cells staying in suspension from the group of cells; and

(iii) comparing the amount of cells settling out of suspension from the group of cells contacted with the compound being tested to the amount of cells settling out of suspension from a control group of cells or comparing the amount of cells staying in suspension from the group of cells contacted with the compound being tested to the amount of cells staying in suspension from a control group of cells,

wherein a difference in the amount of cells settling out of suspension from the group of cells contacted with the compound being tested as compared to the amount of cells settling out of suspension from the control group of cells or difference in the amount of cells staying in suspension from the group of cells contacted with the compound being tested as compared to the amount of cells staying in suspension from the control group of cells identifies the compound as a modulator of TAKl .

26. The method of claim 25, wherein the group of cells, or the control group of cells, or each group is a group of yeast cells .

27. The method of claim 26, wherein the yeast cells are elmlΔ or sak.lΔtσs3ΔelmlΔ mutants.

28. The method of any one of claims 26-27, wherein the yeast cells overexpress TAKl.

29. The method of any one of claims 26-27, wherein the yeast cells express low levels of TAKl.

30. The method of any one of claims 26-29, wherein the yeast cells also express a known activator of TAKl .

31. The method of claim 30, wherein the TAKl activator is TABl, TAB2, TAB3, both TABl and TAB2 or both TABl and TAB3.

32. The method of any one of claims 25-31, wherein the control group of cells is contacted with a known TAKl inhibitor.

33. The method of claim 32 , wherein the TAKl inhibitor is radicicol .

34. The method of claim 25, wherein an increase in the amount of cells settling out of suspension from the group of cells contacted with the compound being tested as compared to the amount of cells settling out of suspension from the control group of cells identifies the compound being tested as an inhibitor of TAKl .

35. The method of claim 25, wherein a decrease in the amount of cells settling out of suspension in the cells contacted with the compound being tested is greater than the amount of cells settling out of suspension in the control cells identifies the compound being tested as an activator of TAKl .

36. A method for detecting transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising:

(i) providing a sample comprising TAKl;

(ii) contacting said sample with a TAKl substrate kinase under conditions permitting phosphorylation of the substrate by TAKl; and

(iii) determining if a phosphate is incorporated into the TAKl substrate kinase wherein the incorporation of phosphate into the TAKl substrate kinase indicates TAKl activity.

37. The method of claim 36, wherein the substrate kinase is or is derived from AMPK.

38. The method of claim 37, ^' wherein the AMPK comprises heterotrimeric AMPK.

39. The method of claim 37, wherein the AMPK comprises mouse AMPK, rat AMPK or human AMPK.

40. The method of claims 36-39, wherein incorporation of the phosphate into AMPK is determined by quantitating the activity of the AMPK.

41. The method of claim 40, wherein AMPK activity is quantified by incorporation of a phosphate into a SAMS peptide .

42. The method of claims 36-39, wherein incorporation of phosphate into AMPK is determined by phosphorylation of T-172 of AMPK.

43. The method of claim 36-42, further comprising quantifying the amount of phosphate incorporated per mol of AMPK, wherein the phosphate level in AMPK indicates the level of TAKl activity present in the sample.

44. A method for identifying a compound as a modulator of transforming growth factor-β-activated protein kinase-1 (TAKl) activity comprising

(i) providing a first sample comprising a TAKl and second sample comprising a TAKl,-

(ii) contacting the first sample with the compound to be tested as a modulator of TAKl activity; (iii) contacting each of the first and second samples with ' a substrate of TAKl under conditions permitting phosphorylation of the substrate; (iv) quantitating the incorporation of a phosphate into the substrate in the first and second samples; and

comparing the incorporation of phosphate into the substrate in the first sample with the incorporation of phosphate into the second sample, wherein a difference in the incorporation of phosphate into the substrate between the first and second samples identifies the compound as a modulator of TAKl activity.

45. A method claim 44, wherein substrate is a substrate kinase.

46. The method of claim 45, wherein the substrate kinase is or is derived from AMPK-

47. A method of increasing AMPK activity comprising contacting AMPK with an agent which increases the activity of TAK in an amount effective to increase AMPK activity.

48. The method of claim 47, wherein AMPK activity is increased by AMPK phosphorylation by TAKl .

49. The method of claim 48, wherein the contacting is performed in vitro.

50. The method of claim 48, wherein the contacting is performed in vivo.

51. The method of claim 47, wherein the agent which increases TAKl activity in the cell is TAKl, an agent -causing an increase in expression of TAKl, causing an increase in expression of TAKl activators, an increase in TAKl associated with TABl and TAB2 , and increase in TAKl associated with TABl and TAB3 , causing downregulation of TAKl inhibitors or causing a decrease in TAKl degradation.

52. A method of treating obesity and diabetes in a subject comprising administering to the subject an agent which activates TAKl in said subject.

53. A method of treating a disease characterized by inflammation in a subject comprising administering to the subject an agent which inhibits TAKl in the subject.

54. The method of claim 53 wherein the disease characterized by inflammation includes arthritis, systemic lupus erythematosus, psoriasis, asthma, inflammatory bowel disease and hypercardiomyotrophy.

55. Use of an agent that increases TAKl activity for the manufacture of a medicament for treating obesity or diabetes in a subject.

56. Use of an agent that inhibits TAKl activity for the manufacture of a medicament for treating a disease characterized by inflammation in a subject.

57. The use of claim 56, wherein the disease characterized by inflammation includes arthritis, systemic lupus erythematosus, psoriasis, asthma, inflammatory bowel disease and hypercardiomyotrophy.