Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.     

Effects of 3-phosphoglycerate and other metabolites on the activation of AMP-activated protein kinase by LKB1-STRAD-MO25

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase (AMPKK). The LKB1-STE-related adaptor (STRAD)-mouse protein 25 (MO25) complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study, we tested an array of metabolites including, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate (3-PG), glucose 1-phosphate, glucose 1,6-bisphosphate, ADP, carnitine, acetylcarnitine, IMP, inosine, and ammonia for allosteric regulation. ADP inhibited both AMPK and LKB1-STRAD-MO25 actions, but probably is not important physiologically because of the low free ADP inside the muscle fiber. We found that 3-PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3-PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3-PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.     

Endurance training increases LKB1 and MO25 protein but not AMP-activated protein kinase kinase activity in skeletal muscle

LKB1 complexed with MO25 and STRAD has been identified as an AMP-activated protein kinase kinase (AMPKK). We measured relative LKB1 protein abundance and AMPKK activity in liver (LV), heart (HT), soleus (SO), red quadriceps (RQ), and white quadriceps (WQ) from sedentary and endurance-trained rats. We examined trained RQ for altered levels of MO25 protein and LKB1, STRAD, and MO25 mRNA. LKB1 protein levels normalized to HT (1 +/- 0.03) were LV (0.50 +/- 0.03), SO (0.28 +/- 0.02), RQ (0.32 +/- 0.01), and WQ (0.12 +/- 0.03). AMPKK activities in nanomoles per gram per minute were HT (79 +/- 6), LV (220 +/- 9), SO (22 +/- 2), RQ (29 +/- 2), and WQ (42 +/- 4). Training increased LKB1 protein in SO, RQ, and WQ (P < 0.05). LKB1 protein levels after training (%controls) were SO (158 +/- 17), RQ (316 +/- 17), WQ (191 +/- 27), HT (106 +/- 2), and LV (104 +/- 7). MO25 protein after training (%controls) was 595 +/- 71. Training did not affect AMPKK activity. MO25 but not LKB1 or STRAD mRNA increased with training (P < 0.05). Trained values (%controls) were MO25 (164 +/- 22), LKB1 (120 +/- 16), and STRAD (112 +/- 17). LKB1 protein content strongly correlated (r = 0.93) with citrate synthase activity in skeletal muscle (P < 0.05). In conclusion, endurance training markedly increased skeletal muscle LKB1 and MO25 protein without increasing AMPKK activity. LKB1 may be playing multiple roles in skeletal muscle adaptation to endurance training.     

Hydrogen sulfide mitigates hyperglycemic remodeling via liver kinase B1-adenosine monophosphate-activated protein kinase signaling

Hyperglycemia (HG) reduces AMPK activation leading to impaired autophagy and matrix accumulation. Hydrogen sulfide (H₂S) treatment improves HG-induced renovascular remodeling however, its mechanism remains unclear. Activation of LKB1 by the formation of heterotrimeric complex with STRAD and MO25 is known to activate AMPK. We hypothesized that in HG; H₂S induces autophagy and modulates matrix synthesis through AMPK-dependent LKB1/STRAD/MO25 complex formation. To address this hypothesis, mouse glomerular endothelial cells were treated with normal and high glucose in the absence or presence of sodium hydrogen sulfide (NaHS), an H₂S donor. HG decreased the expression of H₂S regulating enzymes CBS and CSE, and autophagy markers Atg5, Atg7, Atg3 and LC3B/A ratio. HG increased galectin-3 and periostin, markers of matrix accumulation. Treatment with NaHS to HG cells increased LKB1/STRAD/MO25 formation and AMPK phosphorylation. Silencing the encoded genes confirmed complex formation under normoglycemia. H₂S-mediated AMPK activation in HG was associated with upregulation of autophagy and diminished matrix accumulation. We conclude that H₂S mitigates adverse remodeling in HG by induction of autophagy and regulation of matrix metabolism through LKB1/STRAD/MO25 dependent pathway.     

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.     

Characterization of the liver kinase B1-mouse protein-25 -Ste-20-related adaptor protein complex in adult mouse skeletal muscle

In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.     

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 in vivo. 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 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/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 regulation.     

Diet-induced obese rats exhibit impaired LKB1-AMPK signaling in hypothalamus and adipose tissue

AMPK not only acts as a sensor of cellular energy status but also plays a critical role in the energy balance of the body. In this study, LKB1-AMPK signaling was investigated in diet-induced obese (DIO) and diet resistant (DR) rats. In hypothalamus, DIO rats had lower level of LKB1, AMPKα and pAMPKα than chow-fed or DR rats. Both orexigenic peptide NPY and anorexigenic peptide POMC expression were reduced in hypothalamus of DIO rats. i.c.v. injection of AICAR, an activator of AMPK, increased NPY expression but did not alter POMC expression in DIO rats. In periphery, LKB1 protein content and pAMPKα level were lower in the adipose tissue of DIO rats compared to chow-fed and DR rats. Moreover, pAMPKα and LKB1 protein levels obtained from epididymal fat pad were inversely correlated with epididymal fat mass. LKB1 protein content and pAMPKα in skeletal muscle of DIO rats were not different from those in the muscles of chow-fed and DR rats. In summary, DIO rats, but not DR rats, have impaired LKB1-AMPK signaling in hypothalamus and adipose tissue, suggesting the disturbed energy balance observed in DIO rats is related with abnormalities of AMPK signaling in a tissue specific manner.     

Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate

Recent work has shown that the LKB1 tumour suppressor protein kinase phosphorylates and activates protein kinases belonging to the AMP activated kinase (AMPK) subfamily. In this study, we identify the sucrose non-fermenting protein (SNF1)-related kinase (SNRK), a largely unstudied AMPK subfamily member, as a novel substrate for LKB1. We demonstrate that LKB1 activates SNRK by phosphorylating the T-loop residue (Thr173), and that the LKB1 regulatory subunits STRAD and MO25 are required for LKB1 to activate SNRK. We find that SNRK is not active when expressed in HeLa cells that lack expression of LKB1, and its activity is restored by expression of wild type LKB1, but not catalytically deficient LKB1. We also present evidence that two other AMPK-related kinases more distantly related to AMPK than SNRK, namely NIM1 and testis-specific serine/threonine kinase-1 (TSSK1) are not substrates for LKB1. Tissue distribution analysis indicates that SNRK protein is mainly expressed in testis, similar to TSSK isoforms, whereas NIM1 is more widely expressed. These results provide evidence that SNRK could mediate some of the physiological effects of LKB1.     

Insights into the structural dynamics of Liver kinase B1 (LKB1) by the binding of STe20 Related Adapterα (STRADα) and Mouse protein 25α (MO25α) co-activators

LKB1, the tumour suppressor, is found mutated in Peutz-Jeghers syndrome (PJS). The LKB1 is a serine-threonine kinase protein that is allosterically activated by the binding of STRADα and MO25α without phosphorylating the Thr212 present at activation loop. The present study aims to highlight the structural dynamics and complexation mechanism during the allosteric activation of LKB1 by these co-activators using molecular dynamics simulations. The all atom simulations performed on the complexes of LKB1 with ATP, STRADα, and MO25α for a period of 30 ns reveal that binding of STRADα and MO25α significantly stabilizes the highly flexible regions of LKB1 such as ATP binding region (β1-β2 loop), catalytic & activation loop segments and αG helix. Also, binding of STRADα and MO25α to LKB1 promotes coordinated motion between N- and C-lobes along with the catalytic & activation loops by forming H-bonds between LKB1 and co-activators, which further facilitate to establish the conserved attributes of active LKB1 such as (i) formation of salt bridge between Lys78 and Glu98, (ii) formation of stable hydrophobic R- and C-spines, and (iii) interaction between both catalytic and activation loops. Especially, the residues of LKB1 interacting with STRADα (Arg74, Glu342) and MO25α (Glu165, Pro203 and Phe204) are observed to play a significant role in stabilizing the (LKB1-ATP)-(STRADα-ATP)-MO25α complex. Overall, the present work highlighting the structural dynamics of LKB1 by the binding of allosteric co-activators is expected to provide a basic understanding on drug design specific to PJS syndrome.     

Crystal structure of MO25 alpha in complex with the C terminus of the pseudo kinase STE20-related adaptor

Mouse protein 25 alpha (MO25 alpha) is a 40-kDa protein that, together with the STE20-related adaptor-alpha (STRAD alpha) pseudo kinase, forms a regulatory complex capable of stimulating the activity of the LKB1 tumor suppressor protein kinase. The latter is mutated in the inherited Peutz-Jeghers cancer syndrome (PJS). MO25 alpha binds directly to a conserved Trp-Glu-Phe sequence at the STRAD alpha C terminus, markedly enhancing binding of STRAD alpha to LKB1 and increasing LKB1 catalytic activity. The MO25 alpha crystal structure reveals a helical repeat fold, distantly related to the Armadillo proteins. A complex with the STRAD alpha peptide reveals a hydrophobic pocket that is involved in a unique and specific interaction with the Trp-Glu-Phe motif, further supported by mutagenesis studies. The data represent a first step toward structural analysis of the LKB1-STRAD-MO25 complex, and suggests that MO25 alpha is a scaffold protein to which other regions of STRAD-LKB1, cellular LKB1 substrates or regulatory components could bind.     

Micro-RNA-195 and -451 Regulate the LKB1/AMPK Signaling Axis by Targeting MO25

Background

Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress.

Methodology/Principal Findings

Using a mouse model of hypertrophic cardiomyopathy (HCM), we demonstrated early elevation of miR-195 and miR-451 in HCM hearts, which targets MO25, a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK. We show functional targeting of MO25 by miR-195 and -451. Further in vitro interrogation of MO25 as a functional target validated this hypothesis where over-expression of miR-195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase [ACC] and AMPK activity assay), similar to MO25 knockdown in C2C12 cells by siRNA. Parallel changes were measured in 60 day R403Q HCM male hearts that were rescued by short-term administration of AICAR, an AMPK agonist.

Conclusions/Significance

Elevated miR-195 targets the LKB1/AMPK signaling axis in HCM progression and implicates a functional role in HCM disease progression. MiR-195 may serve as potential therapeutics or therapeutic targets for heart disease.     

MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm

Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADalpha, a catalytically deficient pseudokinase. Here we show that endogenous LKB1-STRADalpha complex is associated with a protein of unknown function, termed MO25alpha, through the interaction of MO25alpha with the last three residues of STRADalpha. MO25alpha and STRADalpha anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25alpha enhances the formation of the LKB1-STRADalpha complex in vivo, stimulating the catalytic activity of LKB1 approximately 10-fold. We demonstrate that the related STRADbeta and MO25beta isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1-STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.     

AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.     

Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction

Recent studies indicate that the LKB1 tumour suppressor protein kinase is the major "upstream" activator of the energy sensor AMP-activated protein kinase (AMPK). We have used mice in which LKB1 is expressed at only approximately 10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 had significantly reduced phosphorylation and activation of AMPKalpha2. In LKB1-lacking muscle, the basal activity of the AMPKalpha2 isoform was greatly reduced and was not increased by the AMP-mimetic agent, 5-aminoimidazole-4-carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase-2, a downstream target of AMPK, was profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, was inhibited in the absence of LKB1. Contraction increased the AMP:ATP ratio to a greater extent in LKB1-deficient muscles than in LKB1-expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin.     

Characterization of an alternative splice variant of LKB1

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.     

Isolation of profilin from embryonic chicken skeletal muscle and evaluation of its interaction with different actin isoforms

An actin-binding protein of 16 kDa was isolated from embryonic chicken skeletal muscle. The protein had the same properties as profilin, exhibited a much higher affinity for cytoskeletal (beta- and gamma-) actins than for sarcomeric (alpha-) actin in the embryonic muscle, and inhibited the polymerization of beta- and gamma-actins more efficiently in a physiological salt solution. These results indicate that the assembly of cytoskeletal and sarcomeric actins is regulated differently by profilin in the developing skeletal muscle, and that the former may not be involved in myofibril assembly.     

MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases

Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25α and MO25β associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25α. MO25α/β binding to MST3/MST4/YSK1 also stimulated kinase activity three- to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.