The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by b-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormonesensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKa1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKa1 by LKB1 in response to lipolytic signals. Activation of AMPKa1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKa1 in vitro. Functional analysis of an AMPKa1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKAactivating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.

Acid secretion in epithelial cells is actively regulated by environmental signals, although the mechanisms by which these cues are translated into activation of H ϩ transport pathways remains the subject of intense research (11,(15)(16)(17)(18)(19). For example, the V-ATPase is regulated by several pathways, which involve CO 2 , phosphatidylinositol 3-kinase, aldolase, phosphofructokinase, actin, microtubules, and angiotensin in a variety of mammalian cellular systems (20 -27). The number of VATPases at the apical membrane of intercalated cells in the kidney increases rapidly under conditions of systemic acidosis (28, 29). Acidosis also induces H ϩ secretion via the V-ATPase through changes in intracellular [Ca 2ϩ ] concentration, calmodulin activation, the cytoskeleton, and by altering the rate of endocytosis and exocytosis in kidney cells (30).We and others have shown that regulation of the V-ATPase at the apical membrane of intercalated and clear cells is tightly linked to alkaline luminal pH, HCO 3 Ϫ , carbonic anhydrase activity, activation of the soluble adenylyl cyclase (sAC),

Transforming growth factor-␤-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Coexpression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain ␣G-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitopetagged TAK1 from transfected cells and mutation of hydrophobic ␣G-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of ␣G-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.Transforming growth factor-␤-activated kinase 1 (TAK1) was first identified as an MAP3K 4 involved in transforming growth factor-␤ and bone morphogenetic protein-mediated signaling (1). Because it transduces a multitude of extracellular stimuli, such as those from interleukin-1, tumor necrosis factor-␣, and lipopolysaccharides, the serine/threonine kinase TAK1 represents a key activator of pathways involving IB kinase, c-Jun NH 2 -terminal kinase (JNK), and p38 (2-9). Furthermore, a regulatory function of TAK1 has also been described in the Wnt signaling pathway (10, 11). TAK1 activity is regulated by association of TAK1-binding proteins, namely TAB1, TAB2, TAB3, and TAB4 (12-17). TAB1, a "pseudophosphatase" that shows close structural similarity with the Mg 2ϩ -or Mn 2ϩ -dependent protein phosphatase family member protein phosphatase 2C␣ (PP2C␣) (18), was originally found in a yeast two-hybrid screen as a protein, which directly triggers TAK1 activity (13). Both structural and functional studies revealed that an evolutionarily conserved motif in the carboxyl-terminal region of TAB1 (residues 480 -504) is sufficient to bind to the catalytic domain of TAK1. However, the presence of a slightly extended TAB1 carboxyl-terminal domain (residues 437-504) is indispensable for full activation of TAK1 in HeLa cells (19 -2...

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na ϩ -dependent creatine uptake into CRTexpressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmax parameter. [ 14 C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-␤-Dribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranosyl 5=-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress. proximal tubule; metabolism; Xenopus oocytes; target of rapamycin; SLC6A8 AMP-ACTIVATED PROTEIN KINASE (AMPK) is a ubiquitous metabolic-sensing kinase that exists as an ␣,␤,␥ heterotrimer and is activated by cellular energy depletion (elevated ratios of intracellular AMP to ATP concentration) and other cellular stresses (e.g., Ca 2ϩ stress). AMPK activation involves phosphorylation of the catalytic ␣-subunit at Thr 172 (pThr 172 ) by upstream AMPK kinases that include the LKB1 complex and the Ca 2ϩ

(28 -30, 49). We have demonstrated that in kidney intercalated cells and in epididymal clear cells, which share a common developmental origin in the Wolffian duct, the V-ATPase redistributes from the apical membrane to the cytosol with stimulation of the metabolic sensor AMPK (16,18). In our previous studies we first identified the V-ATPase A subunit as an AMPK substrate using the unbiased "MudSeeK" proteomic screening method and subsequently showed that the V-ATPase A subunit is phosphorylated directly by AMPK in kidney cells (18,52). AMPK is an important cell energy-sensing kinase that has been shown to downregulate several kidney membrane transport proteins (17,39). We have shown that AMPK reduces apical V-ATPase accumulation acutely in kidney intercalated cells, and that AMPK activation antagonizes the ability of PKA to increase V-ATPase localization at the apical pole of type A intercalated cells (16).Phosphatidylinositol 3-kinase and PKA are additional kinases that regulate the function of the V-ATPase in the mammalian kidney (16,41,43). PKA agonists also regulate the V-ATPase in other animal models (42, 54). However, it was not until recently that work by our group linked the direct phosphorylation of the V-ATPase by PKA at Ser-175 to increases in V-ATPase plasma membrane activity in mammalian cells (2). Ser-175 A subunit phosphorylation is likely to occur downstream of acid-base-sensing pathways, which require the presence of active carbonic anhydrase, the soluble