Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 invivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25α can be expressed in soluble form, whereas the other two, LKB1 and STRADα are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25α-STRADα complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25α and STRADα exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADα, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the α-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the α-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulatio

An automated home-built low-cost fermenter suitable for large-scale bacterial expression of proteins in Escherichia coli.

International audienceWe have developed an automated fermentation system for cost-efficient upscaling of protein expression in bacteria. The system, built for use by nonbiotechnologists, can be assembled mostly from standard laboratory equipment and allows a largely unattended growth of bacteria to OD 25 (at 600 nm) in a 12 L vessel. The typical yield of 250-350 g of wet weight cell pellet per run, which is equivalent to the biomass obtained from 250 shake flask cultures containing 400 mL Luria-Broth medium each, facilitates the production of large amounts of purified recombinant protein without the laborious need for optimization of expression and purification conditions

Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex

ISSN:1073-6085ISSN:1559-030

Dissecting the role of 5'-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase.

International audienceAMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK alpha1beta1gamma1 and alpha2beta2gamma1 by their upstream kinases (Ca(2+)/calmodulin-dependent protein kinase kinase-beta and LKB1-MO25alpha-STRADalpha), the deactivation by protein phosphatase 2Calpha, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 mumol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 microm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK alpha-subunits at Thr-172 by protein phosphatase 2Calpha is attenuated by AMP. Furthermore, it is shown that neither purified NAD(+) nor NADH alters the activity of AMPK in a concentration range of 0-300 microm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation

The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins

beta1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β1Pix knockdown, or overexpression of WT β1Pix or the β1Pix-Δ602-611 mutant. Moreover, we found that β1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction, phosphorylation and ER localization

AbstractAMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping

PKA phosphorylates and inactivates AMPKalpha to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive 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 AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 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 PKA-activating 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