Dr Andrew C Pearce
email: a.c.pearce@bham.ac.uk

Owen Jt Mccarty

Simon Dj Calaminus

Elena Vigorito

Martin Turner

Steve P Watson

Owen J T Mccarty

Simon D J Calaminus

Vav family proteins are

Keywords: Vav, platelet spreading, integrin IIb 3, signalling, PLC 2, fibrinogen

come

Introduction

Platelets are small anucleate cells that circulate in a quiescent state in the vasculature.

Following vascular damage, platelets are recruited to the site of injury and undergo explosive activation to stem the loss of blood from the wound. Following tethering, platelets are activated by prolonged close proximity of the platelet immunoglobulin receptor GPVI with subendothelial collagen (reviewed in [START_REF] Nieswandt | Platelet-collagen interaction: is GPVI the central receptor?[END_REF]). GPVI stimulates an increase in the affinity of the major platelet integrin IIb 3 for its ligands, von Willebrand factor (VWF) and fibrinogen. Outside-in signalling by the integrin contributes to spreading over the subendothelial matrix and recruitment of additional platelets which become crosslinked by fibrinogen, thereby generating a vascular plug [START_REF] Nesbitt | Intercellular calcium communication regulates platelet aggregation and thrombus growth[END_REF]. Finally, the clot is retracted and stabilised by an active process dependent on IIb 3 and the cytoskeleton [START_REF] Law | Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function[END_REF].

The Vav family of GTP exchange factors (GEFs) consists of three members [START_REF] Katzav | vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells[END_REF][START_REF] Schuebel | Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes[END_REF][START_REF] Movilla | Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins[END_REF][START_REF] Henske | Identification of VAV2 on 9q34 and its exclusion as the tuberous sclerosis gene TSC1[END_REF] which share a common structural arrangement. The amino terminus contains a calponin homology domain and an acidic region, which contains regulatory tyrosine phosphorylation sites. This is followed by Dbl homology, pleckstrin homology (PH) and zinc finger domains, which form the GDP/GTP exchange factor region of Vav family proteins. The C terminal portion contains a short proline rich region and an SH3-SH2-SH3 region. Vav2 and Vav3 are widely expressed, whereas Vav1 is specifically expressed in haematopoietic cells [START_REF] Schuebel | Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes[END_REF][START_REF] Movilla | Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins[END_REF][START_REF] Henske | Identification of VAV2 on 9q34 and its exclusion as the tuberous sclerosis gene TSC1[END_REF]. The guanine nucleotide exchange activity of Vav proteins is specific for the Rho family of small G proteins. The individual Vav proteins have specificity towards different Rho family G proteins. For example, Vav1 selectively activates Rac1, Rac2 and RhoG, whereas Vav2 and Vav3 show less activity towards Rac1 [START_REF] Movilla | Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins[END_REF][START_REF] Schuebel | Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2[END_REF]. The GTP/GDP exchange activity of all three Vav proteins is modulated through tyrosine phosphorylation of regulatory tyrosine residues by Src and Syk family kinases [START_REF] Bustelo | Regulatory and signaling properties of the Vav family[END_REF].

Vav proteins are prominent tyrosine kinase substrates downstream of ITAM receptors, including the platelet collagen receptor GPVI, and the T-and B-cell antigen receptors [START_REF] Movilla | Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins[END_REF][START_REF] Bustelo | Regulatory and signaling properties of the Vav family[END_REF][START_REF] Pearce | Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C[END_REF][START_REF] Pearce | Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen[END_REF][START_REF] Turner | VAV proteins as signal integrators for multi-subunit immune-recognition receptors[END_REF] FITC-phalloidin and Oregon Green Bapta1-AM were from Molecular Probes (Leiden, The Netherlands). All other reagents were purchased from Sigma (Poole, UK) or obtained from previously described sources [START_REF] Poole | The Fc receptor gammachain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen[END_REF][START_REF] Pearce | The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa[END_REF].

Animals:

The generation of mice disrupted in the vav1 gene (Vav1 -/-) is described in Turner et al. [START_REF] Turner | A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes[END_REF]. The generation of mice disrupted in the vav2 gene (Vav2 -/-) is described in Doody et. al. [START_REF] Doody | Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation[END_REF].The generation of mice disrupted in the vav3 gene (Vav3 -/-) is described in Fujikawa et al. [20]. Compound knockout mice were generated by appropriate crossing of the individual knockout genotypes. Mutant and control mice were age and background matched. All animals were maintained using housing and husbandry in accordance with local and national legal regulations.

Preparation of mouse platelets: Blood was taken from a terminally CO 2 -narcosed mouse by cardiac puncture on the day of the experiment into 1:10 (v:v) ACD (85 mM sodium citrate, 110 mM glucose, 71 mM citric acid). Blood was diluted 1:6 (v:v) in Tyrodes-HEPES buffer (134 mM NaCl, 0.34 mM Na 2 HPO 4 , 2.9 mM KCl, 12 mM NaHCO 3 , 20 mM HEPES, 5 mM glucose, 1 mM MgCl 2 , pH 7.3) and centrifuged at 200g to obtain platelet rich plasma (PRP). PRP was centrifuged in the presence of 0.1µg/ml prostacyclin at 1000g and the platelet pellet was resuspended in Tyrodes-

HEPES.

Flow cytometry staining: Washed platelets (1x10 7 /ml) were stained with the indicated FITC-conjugated antibodies for 15 minutes at room temperature. Staining was quenched with 400 l tyrodes-HEPES buffer and samples were analysed using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, UK).

Static adhesion spreading assay:

Glass coverslips were coated with 100 µg/ml fibrinogen solution overnight at 4 o C followed by washing with PBS. Slides were then blocked using 0.5% heat denatured BSA in PBS for 1 hour at room temperature, followed by washing in PBS. Mouse platelets, resuspended at a concentration of 3 x 10 7 platelets/ml in Tyrodes-HEPES containing 2U/ml apyrase and 10µM indomethacin, were transferred to the coverslips and incubated at 37 o C for 45 min in a humid atmosphere. Excess platelets were removed and the adherent platelets were fixed with 3.7% paraformaldehyde for 10 min at room temperature. The coverslips were washed in PBS, mounted using HydroMount Mounting Medium (National Diagnostics, Hull, UK) and viewed under differential interference contrast microscopy (DIC) under a 63x oil immersion lens and Slidebook software (Intelligent Imaging Innovations, Colorado, USA). Surface areas were calculated using a Java plug in for ImageJ software.

Immunoprecipitation and immunoblotting: Platelets at 2x10 8 /ml were incubated over fibrinogen-coated dishes for 45 minutes at 37°C in the presence of 2U/ml apyrase and 10 M indomethacin. Non-adherent basal platelets were removed and lysed in an equal volume of 2x lysis buffer (2% NP-40, 300 mM NaCl, 20 mM Tris, 2 mM EDTA, 2mM EGTA, 2 mM Na 3 VO 4 , 200 g/ml AEBSF hydrochloride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µg/ml pepstatin A, pH 7.4). Adherent platelets were lysed in a final volume of 1ml 1x lysis buffer. Insoluble cell debris was removed by centrifugation for 5 min at 13,000g, 4 C and cell lysates were precleared using protein A-Sepharose. Platelet lysates were incubated with the indicated primary antibodies and resulting protein complexes and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed as described previously [START_REF] Pearce | Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen[END_REF] with detection by enhanced chemiluminescence (ECL, Amersham Biosciences, Bucks, UK).

Rac activation assay: Rac activity was measured as described previously [START_REF] Pearce | Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C[END_REF]. The CRIB domain of PAK1 (amino acids 67-150) was expressed as a GST fusion protein and bound to glutathione sepharose beads. Platelet stimulations were stopped with an equal volume of 2x Rac assay lysis buffer (2% (v:v) NP-40, 1% (w:v) N-octyl glucoside, 300 mM NaCl, 20 mM Tris, 2 mM EDTA, 2mM EGTA, 20 mM MgCl 2, 2 mM Na 3 VO 4 , 200 g/ml AEBSF hydrochloride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µg/ml pepstatin A, pH 7.4). Insoluble material was removed by centrifugation (5 minutes, 13000 rpm, 4°C) and freshly prepared GST-PAK1 was added to the samples and incubated for 1 hour at 4°C. The beads were then washed with 1x Rac assay lysis buffer and the bound protein taken up into Laemmli buffer.

The resulting samples were separated by 12% SDS-PAGE and transferred to PVDF membranes for immunoblotting as described above.

F-actin assay: Filamentous actin was measured using a modification of the method of Machesky and Hall as previously described [54]. Briefly, platelets at a concentration of 2x10 8 /ml were fixed with an equal volume of 3.7% formaldehyde containing FITCphalloidin (20 mM KH 2 PO 4 , 10 mM Pipes, 5 mM EGTA, 2 mM MgCl 2 , 0.1% Triton X-100, 3.7% formaldehyde, 2 M FITC-phalloidin) and rotated for 1 hour at room temperature. Platelets were then pelleted for 2 minutes in a microcentrifuge and washed in 0.1% saponin, 20 mM KH 2 PO 4 , 10 mM Pipes, 5 mM EGTA, 2 mM MgCl 2 .

Platelets were rotated in methanol for 1 hour to extract the FITC-phalloidin. FITCphalloidin binding was measured by measuring the emission at 520 nm using an excitation wavelength of 488 nm on a spectrofluorimeter. Ca 2+ mobilisation assay: Platelets were incubated with 15 M Oregon Green BAPTA 1-AM in DMSO containing 1.2mg/ml pluronic acid for 90 minutes in the dark at room temperature. Excess dye was removed by washing the platelets in Tyrodes-HEPES buffer in the presence of 0.1µg/ml prostacyclin. Platelets were allowed to settle onto fibrinogen-coated coverslips in the presence of 2U/ml apyrase and 10 M indomethacin and imaged in real time using the 63x oil immersion lens on a Zeiss Axiovert 200M microscope using Slidebook software (Intelligent Imaging Innovations, Colorado, USA).

Results

Vav1/3 -/-platelets exhibit reduced spreading on fibrinogen

To investigate whether Vav family proteins are involved in outside-in signalling by IIb 3, platelets from wild type and Vav1/3 -/-mice were incubated on fibrinogencoated coverslips for 45 minutes and imaged by DIC microscopy. These experiments were carried out in the presence of apyrase and indomethacin to block the effects of the secreted G protein-coupled secondary agonists, ADP and thromboxane A 2 . Under these conditions, wild type mouse platelets undergo partial spreading with formation of filopodia and limited lamellipodia-like structures (Figure 1A). This limited spreading response has previously been shown to be independent of activation of Rac, as shown using Rac1/Rac2-deficient platelets [START_REF] Mccarty | Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow[END_REF], but dependent on PLC 2 [START_REF] Wonerow | A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading[END_REF].

Vav1/3-deficient platelets adhere with the same efficiency as wild type platelets (Figure 1B). However, in contrast to wild type platelets, Vav1/3 -/-platelets do not form lamellipodia and have less filopodia than wild type cells (Figure 1A). The number of filopodia formed by wild type and Vav1/3-deficient platelets were counted and plotted as a bar chart. Vav1/3-deficient platelets exhibit significantly less filopodia than wild type cells (Figure 1C). The surface area of each set of platelets was measured and plotted as a frequency distribution. The distribution of surface area of Vav1/3 -/-platelets is shifted slightly to the left relative to wild type platelets (Figure 1D), thereby demonstrating a small, but significant reduction in surface area as shown in Table 1. Platelets deficient in Vav1 or Vav3 alone exhibited an intermediate phenotype with a slightly lower average surface area than wild type platelets (Table 1A). The surface area of Vav1/3 -/-platelets was significantly reduced relative to the single knockouts demonstrating a degree of redundancy in the role of these proteins in platelet spreading on fibrinogen (Table 1A). Consistent with a residual expression of Vav2 in platelets, the defect in spreading in Vav1/2/3-deficient platelets was not significantly different to that in Vav1/3-deficient platelets (Table 1A).

A potential explanation for the decreased spreading of Vav1/3 -/-platelets on fibrinogen could be due to reduced surface expression of the fibrinogen receptor IIb 3 in these cells. To investigate this, we have compared the levels of the integrin on wild type and Vav1/3-deficient platelets by staining with antibodies to each of the subunits of the integrin and to the integrin complex. Expression of IIb, 3 and IIb 3 is identical between wild type and Vav1/3-deficient platelets (Figure 1E).

These results demonstrate that the defective spreading of Vav1/3-deficient platelets on fibrinogen is not due to a lower expression level of the receptor.

Activation of PLC 2 by IIb 3 is impaired in Vav1/3 -/-platelets Platelet spreading on fibrinogen has previously been shown to be critically dependent on Ca 2+ , PKC and PLC 2 [START_REF] Wonerow | A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading[END_REF][START_REF] Barkalow | Alpha-adducin dissociates from F-actin and spectrin during platelet activation[END_REF]. Since Vav family proteins have been shown to be critical for regulation of PLC 2 by ITAM receptors in platelets and in other cells, we investigated whether a defect in PLC 2 regulation by the integrin could underlie the reduced spreading on fibrinogen that is observed in the combined absence of Vav1 and Vav3. Wild type and Vav1/3 -/-platelets were incubated over a fibrinogen-coated surface for 45 min, before removal and lysis of basal (non-adherent) and stimulated (adherent) platelets. Following lysis and immunoprecipitation, PLC 2 phosphorylation was measured by western blotting using the antiphosphotyrosine antibody, 4G10. Wild type platelets exhibit a robust increase in tyrosine phosphorylation of PLC 2 when spread on fibrinogen, which is reduced by approximately 50% in Vav1/3 -/-platelets (Figure 2A). In contrast, tyrosine phosphorylation of the upstream kinase Syk is not significantly different in wild type and Vav1/3 -/-platelets (Figure 2A). These results suggest that the same amount of signal is transmitted by the receptor into the cell but that the efficiency of transmitting this signal to PLC 2 is reduced in the absence of Vav1/3.

In order to investigate if the defect in platelet spreading is due to the defect in signalling of IIb 3 to PLC 2, we assessed the ability of Vav1/3 -/-platelets to mobilise Ca 2+ during spreading on fibrinogen. Platelets from wild type and Vav1/3deficient mice were labelled with the Ca 2+ reporter dye Oregon Green BAPTA 1-AM. This dye is a highly sensitive Ca 2+ reporter dye that can be used to detect fluctuations in intracellular Ca 2+ levels without chelating sufficient Ca 2+ to block Ca 2+ -dependent functional responses (data not shown and [START_REF] Yap | Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets and transfected Chinese hamster ovary cells[END_REF][START_REF] Yuan | The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells[END_REF]). Labelled platelets were incubated over fibrinogen-coated coverslips and spreading was monitored by fluorescence microscopy. Wild type platelets exhibit a series of oscillations in fluorescence, indicative of transient elevation of cytosolic Ca 2+ as shown in Figure 2B and the supplementary video Figure S1A. In contrast, Vav1/3 -/-platelets exhibit significantly less Ca 2+ transients over a seven minute recording period as shown by the single cell records (Figure 2B and supplementary video Figure S1B) and pooled data (Figure 2C). These results demonstrate that Vav1 and Vav3 are required for IIb 3-mediated Ca 2+ mobilisation, regulated downstream of PLC 2.

Vav1 and Vav3 do not regulate Rac in fibrinogen or thrombin stimulated platelets

In addition to their role in regulating PLC 2 downstream of ITAM receptors, Vav family proteins also function as GTP exchange factors for Rho family G proteins. The defect in spreading that is observed in the absence of Vav1 and Vav3 is unlikely to be due to blockade of Rho activity as platelets are not thought to form stress fibres under these conditions. Furthermore, we have been unable to detect activation of Rho in platelets that have adhered to fibrinogen. However, we have also been unable to detect activation of Cdc42 during adhesion of human or murine platelets to fibrinogen (data not shown). This may reflect that the small G protein is not activated by the integrin or that it is activated at a level below that required for detection. We have previously reported that spreading of mouse platelets on fibrinogen is independent of activation of Rac as shown using Rac1/2-deficient mice [START_REF] Mccarty | Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow[END_REF]. Furthermore, we were unable to detect activation of Rac1/2 in human and mouse platelets using GST-PAK, which selectively binds to the GTP-bound form of the protein, to precipitate active Rac [START_REF] Mccarty | Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow[END_REF]. In the present study we have been able to detect weak activation of Rac in murine platelets that have spread on fibrinogen (Figure 3A). In order to investigate if Rac activation by the integrin is regulated by Vav1/3, Rac activation in Vav1/3deficient platelets was assayed. IIb 3 induced Rac activation is normal in Vav1/3 deficient platelets (Figure 3A). These results demonstrate that Vav1/3 does not lie upstream of Rac in IIb 3 signalling.

In contrast to signalling by the integrin, thrombin induces robust activation of Rac. In order to investigate whether Vav1/3 lies upstream of Rac in thrombinstimulated platelets, active Rac was precipitated from thrombin stimulated platelets as above. Activation of Rac by thrombin is not altered in the absence of Vav1 and Vav3 (Figure 3B). Furthermore, thrombin induced F-actin polymerisation is normal in Vav1/3 deficient platelets (Figure 3C). These results demonstrate that Vav1/3 does not lie upstream of Rac or actin assembly in thrombin stimulated platelets.

Importantly this is consistent with the observation that thrombin induces full

Discussion

In the present study we have investigated the role of Vav family proteins in IIb 3 mediated platelet activation and spreading on fibrinogen. Platelets deficient in the major Vav family proteins, Vav1 and Vav3, have reduced spreading on fibrinogen, in association with reduced tyrosine phosphorylation of PLC 2 and reduced elevation of intracellular Ca 2+ . These results therefore demonstrate a critical role for Vav1 and Vav3 in the regulation of PLC 2 by IIb 3, as has previously been shown to be the case for regulation of PLC 2 by the ITAM-coupled collagen receptor, GPVI [START_REF] Pearce | Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen[END_REF]. The defect in spreading in the Vav1/3 -/-platelets is by-passed in the presence of the G protein receptor agonist, thrombin, which stimulates powerful activation of PLC isoforms in platelets. Thus, the role of Vav1 and Vav3 in mediating spreading on fibrinogen is specifically linked to IIb 3-mediated outside-in signalling.

Vav family proteins have been implicated in integrin-dependent responses in a variety of cells [START_REF] Yron | Integrin-dependent tyrosine phosphorylation and growth regulation by Vav[END_REF][START_REF] Gakidis | Vav GEFs are required for beta2 integrin-dependent functions of neutrophils[END_REF][START_REF] Gao | Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes[END_REF]. Vav-deficient neutrophils display defective 2-integrin mediated spreading and phagocytosis [START_REF] Gakidis | Vav GEFs are required for beta2 integrin-dependent functions of neutrophils[END_REF], a K562 model of v 3-mediated cell adhesion demonstrates recruitment of Vav1 to phosphorylated 3-subunit in fibronectin-adherent cells [START_REF] Gao | Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes[END_REF] and CHO cells over-expressing IIb 3, Syk, SLP-76 and Vav1 form extensive lamellipodia on fibrinogen [38]. In the present study, we demonstrate that Vav1 and Vav3 are required for normal platelet spreading on fibrinogen, as a consequence of a reduction in IIb 3-mediated activation of PLC 2 and Ca 2+ mobilisation. Since 2 integrins in neutrophils also exhibit PLC 2 dependent Ca 2+ mobilisation [START_REF] Hellberg | Ca2+ signalling mechanisms of the beta 2 integrin on neutrophils: involvement of phospholipase C gamma 2 and Ins(1,4,5)P3[END_REF], we therefore suspect that Vav proteins are likely to be involved in PLC 2 regulation by 2-integrins in neutrophils and that this contributes to the defective functional responses in these cells [START_REF] Gakidis | Vav GEFs are required for beta2 integrin-dependent functions of neutrophils[END_REF]. Interestingly, platelet responses to the G protein coupled agonist thrombin are not affected by deficiency of Vav1 and Vav3. Similarly, neutrophil responses to G protein coupled chemoattractants are normal in Vav deficient cells suggesting that Vav is also dispensable for GPCR signalling in these cells [START_REF] Gakidis | Vav GEFs are required for beta2 integrin-dependent functions of neutrophils[END_REF].

It is now recognised that integrins use many of the same signalling proteins as ITAM receptors [START_REF] Watson | GPVI and integrin alphaIIb beta3 signaling in platelets[END_REF]. In platelets, for example, critical roles for Src [22,42] and Syk[22] tyrosine kinases, the adapter protein SLP-76 [START_REF] Judd | Differential requirement for LAT and SLP-76 in GPVI versus T cell receptor signaling[END_REF] and PLC 2 [START_REF] Wonerow | A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading[END_REF][START_REF] Goncalves | Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2[END_REF][START_REF] Suzuki-Inoue | Murine GPVI stimulates weak integrin activation in PLCgamma2-/-platelets: involvement of PLCgamma1 and PI3-kinase[END_REF] have been reported downstream of both GPVI and IIb 3. Based on the data presented here, a further similarity in the signalling pathways activated by these two classes of receptors is the requirement of Vav proteins for efficient regulation of PLC 2 [START_REF] Pearce | Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C[END_REF][START_REF] Pearce | Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen[END_REF].

It is noteworthy that Vav proteins appear to be essential for optimal regulation of PLC isoforms by all ITAMs. It will therefore be of interest to know whether Vav proteins are required downstream of all integrins for the regulation of PLC .

Interestingly, we have also recently demonstrated a role for Vav1 and Vav3 in the activation of PLC 2 by the C-type lectin family receptor, CLEC-2, in platelets which has a single rather than dual YXXL motif in its cytosolic tail [START_REF] Suzuki-Inoue | A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2[END_REF]. There are however significant differences in signalling by these three classes of receptor in platelets. For example, GPVI signalling takes place in lipid rafts and is critically dependent on the transmembrane adaptor protein, LAT [61,[START_REF] Wonerow | Differential role of glycolipid-enriched membrane domains in glycoprotein VI-and integrin-mediated phospholipase Cgamma2 regulation in platelets[END_REF], whereas signalling by IIb 3 is independent of these specialised membrane domains and LAT [START_REF] Wonerow | A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading[END_REF][START_REF] Wonerow | Differential role of glycolipid-enriched membrane domains in glycoprotein VI-and integrin-mediated phospholipase Cgamma2 regulation in platelets[END_REF]. In comparison, signalling by CLEC-2 is partially dependent on the adapters LAT and SLP-76, whereas the latter is essential for responses to GPVI.

Cell spreading involves extensive cytoskeletal re-modifications, which can be regulated by Rho family small G proteins. The small G proteins Cdc42, Rac and Rho are implicated in the formation of filopodia, lamellipodia and stress fibres, respectively [START_REF] Nobes | Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia[END_REF][64][START_REF] Ridley | The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors[END_REF]. In the present study we have assayed spreading directly mediated by the integrin, by performing all studies in the presence of apyrase and indomethacin. Under these conditions, mouse platelets undergo a limited spreading response that is dependent on PLC 2 [START_REF] Wonerow | A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading[END_REF] but independent of Rac [START_REF] Mccarty | Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow[END_REF]. Consistent with this, we have been able to detect only weak activation of Rac by IIb 3 in platelets that have undergone spreading on fibrinogen and this activation is not reduced in Vav1/3-deficient platelets. These results demonstrate that the defective regulation of PLC 2 by IIb 3 in platelets is not due to defective regulation of Rac. Interestingly, we have been unable to detect activation of Cdc42 (data not shown) during spreading on fibrinogen despite robust formation of filopodia. This may represent a low level of activation of the small G protein that is below the level of detection or may represent Cdc42 independent formation of filopodia, as has been reported in other cells [START_REF] Czuchra | Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells[END_REF]67]. We can therefore not rule out the possibility that the defect in spreading on fibrinogen in the absence of Vav1 and Vav3 is, at least partially, due to a direct inhibition of Cdc42 activation or a related G protein, although it should be noted that a role of Vav family GTP exchange factors in the regulation of Cdc42 is controversial [START_REF] Movilla | Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins[END_REF][START_REF] Schuebel | Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2[END_REF][68][START_REF] Marignani | Vav2 is required for cell spreading[END_REF][START_REF] Abe | Vav2 is an activator of Cdc42, Rac1, and RhoA[END_REF][START_REF] Movilla | How Vav proteins discriminate the GTPases Rac1 and RhoA from Cdc42[END_REF]. It is also extremely unlikely that the spreading defect of Vav1/3 deficient platelets is due to impairment of activation of Rho as there is no evidence of stress fibre formation in platelets spread under these conditions.

Under physiological conditions the limited spreading response initiated by IIb 3 is reinforced by G protein coupled agonists such as ADP and thrombin leading to Rac activation and Rac-dependent full spreading [START_REF] Pearce | Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C[END_REF][START_REF] Mccarty | Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow[END_REF]. In the present study we have shown that Vav proteins are not required for G protein coupled receptor signalling leading to Rac activation or actin polymerisation, suggesting that this process would occur normally in Vav deficient mice.

It is well established that Vav family proteins have GEF-independent functions thought to be due to their role as adaptor proteins and stabilising signalling complexes [START_REF] Manetz | Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells[END_REF][START_REF] Reynolds | Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3kinase-dependent and -independent pathways[END_REF]27,[START_REF] Doody | Vav-2 controls NFATdependent transcription in B-but not T-lymphocytes[END_REF]. Indeed, Vav family proteins have been shown to be required for normal formation of the LAT-Gads-SLP-76-PLC 2 signalosome downstream of the T cell receptor [START_REF] Reynolds | Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3kinase-dependent and -independent pathways[END_REF]. It is likely that this function of Vav proteins is responsible for their critical role in the regulation of PLC isoforms by ITAM receptors, including GPVI. Demonstration of inhibition of PLC 2 downstream of IIb 3 in the present study provides strong evidence that this mechanism also applies to the regulation of PLC isoforms downstream of integrin receptors.

In summary, the data presented here demonstrate an important role for Vav The surface area of 301 wild type and 319 Vav1/3 -/-platelets on fibrinogen was calculated by imaging slides as above with a graticule standard and using Image J software to measure surface area. Surface areas are plotted as a frequency distribution.

(E) Washed platelets from WT or Vav1/3 -/-mice were stained with FITC-conjugated rat IgG (shaded) or rat anti-mouse IIb, 3 or IIb 3 (non-shaded). Results are from one experiment that is representative of two others. Green BAPTA 1-AM and incubated over fibrinogen-coated coverslips in the presence of 2U/ml apyrase and 10 M indomethacin and imaged in real time using the FITC

  . The importance of Vav in signalling by ITAM receptors has been demonstrated by studying mice that lack individual or combinations of Vav family proteins [13-21]. In all cases, Vav proteins have been shown to play a critical role in the regulation of PLC by ITAM receptors. Vav1 deficient T cells have significantly reduced TCR signalling with a more pronounced phenotype observed in Vav1/3 -/-and Materials and Methods Antibodies and reagents: Anti-phosphotyrosine monoclonal antibody (mAb) 4G10 and anti-Rac mAb were purchased from Upstate Biotechnology (TCS Biologicals Ltd., Bucks, UK). The anti-PLC 2 and anti-Syk pAbs were kindly supplied by Dr. Mike Tomlinson (DNAX, Palo Alto, CA., USA). The FITC-conjugated rat antimouse IIb (MwReg30), 3 (Luc.A5) and IIb 3 (Leo.D2) antibodies and rat IgG were purchased from Emfret Analytics (Wurzburg, Germany). The cDNA for GST-PAK Crib domain was a kind gift from Dr Doreen Cantrell (ICRF, London, UK).

  family proteins in signalling by the platelet integrin IIb 3 and for normal regulation of PLC 2, intracellular Ca 2+ mobilisation and platelet spreading on fibrinogen. These results add to previous studies that demonstrate a pivotal role for Vav family proteins in the regulation of PLC 2 by distinct classes of surface glycoproteins, including integrins, ITAM and lectin receptors. Biochemical Journal Immediate Publication. Published on 23 Oct 2006 as manuscript BJ20061508 Copyright 2006 Biochemical Society

Figure Legends:Figure 1 :

 1 Figure Legends:

Figure 2 :

 2 Figure 2: (A) Washed platelets were incubated over fibrinogen-coated petri dishes in the presence of 2U/ml apyrase and 10 M indomethacin for 45 minutes at 37ºC. Basal (non-adherent) and fibrinogen stimulated (adherent) cells were lysed in NP40 lysis buffer and PLC 2 and Syk were immunoprecipitated. Immunoprecipitates were separated by SDS PAGE and western blotted with 4G10 anti-phosphotyrosine antibody. Membranes were subsequently stripped and reprobed for PLC 2 and Syk respectively. (B) Washed platelets were labelled with the Ca 2+ reporter dye Oregon

Biochemical Journal Immediate Publication. Published on 23 Oct 2006 as manuscript BJ20061508 Copyright 2006 Biochemical Society Analysis of data:

  Experiments were performed on at least 3 occasions and are shown as representative data. Where appropriate data is shown +/-Standard Error. Where statistical significance is indicated data has been subjected to student t test. For comparison of surface areas of platelets in table 1A where multiple comparisons are required, Tukey test has been used. p<0.01 was selected to represent statistical significance.

Biochemical Journal Immediate Publication. Published on 23 Oct 2006 as manuscript BJ20061508 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 23 Oct 2006 as manuscript BJ20061508

Acknowledgements:

The authors gratefully acknowledge Helen Reynolds and SABU staff for animal care and Majd Protty for computational assistance. This work was supported by the Wellcome Trust, the British Heart Foundation and the BBSRC. SPW holds a British Heart Foundation Research Chair and MT holds an MRC Senior Fellowship.


Matthew J Sanders

Pascal O Grondin

Bronwyn D Hegarty

Michael A Snowden

Prof David Carling
email: dcarling@csc.mrc.ac.uk

Investigating the mechanism for AMP activation of the AMPactivated protein kinase cascade

Keywords: AMP-activated protein kinase, Ca 2+ /calmodulin dependent protein kinase kinase, energy metabolism, LKB1, metabolic syndrome, phosphorylation

AMP-activated protein kinase (AMPK) is activated allosterically by AMP and by phosphorylation of threonine residue 172 (T172) within the catalytic subunit (α). Here we show that mutations in the γ regulatory subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of T172 by protein phosphatase 2Cα (PP2Cα). Moreover, a mutation in the γ subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on T172 phosphorylation by either LKB1 or Ca 2+ /calmodulindependent protein kinase kinase (CaMKK) β using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver there was an apparent AMPstimulation of T172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a protein phosphatase inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Cα in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of T172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Cα, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: direct allosteric activation and by protecting T172 from dephosphorylation. Based on our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKKβ. This model accounts for activation of AMPK by two distinct signals: a Ca 2 + -dependent pathway, mediated by CaMKKβ and an AMP-dependent pathway, mediated by LKB1.

INTRODUCTION

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a key role in energy homeostasis [START_REF] Carling | The AMP-activated protein kinase cascade--a unifying system for energy control[END_REF][START_REF] Kahn | AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism[END_REF]. AMPK is a heterotrimeric complex of a catalytic subunit (α) and two regulatory subunits (β and γ). Isoforms of all three subunits have been identified in mammals that allow for the generation of 12 different heterotrimeric complexes [START_REF] Carling | The AMP-activated protein kinase cascade--a unifying system for energy control[END_REF]. The yeast counterpart of AMPK is the SNF1 (sucrose nonfermenting) complex, composed of Snf1 (α), Snf4 (γ) and either Sip1, Sip2 or Gal83 (β isoforms) [START_REF] Hardie | The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?[END_REF]. Both AMPK and SNF1 are activated by phosphorylation by upstream kinases.

In yeast, 3 upstream kinases have been identified corresponding to Sak1 (originally referred to as Pak1), Elm1 and Tos3 [START_REF] Hong | Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases[END_REF][START_REF] Sutherland | Elm1p Is One of Three Upstream Kinases for the Saccharomyces cerevisiae SNF1 Complex[END_REF]. In mammals, LKB1 and CaMKKβ (Ca 2+ -calmodulin dependent protein kinase kinase) have been identified as physiological kinases upstream of AMPK [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF][START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF][START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF][START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress[END_REF][START_REF] Hurley | The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases[END_REF]. More recently, evidence has been reported indicating that Tak1, a member of the mitogen activated protein kinase kinase kinase family, is also capable of phosphorylating and activating AMPK [START_REF] Momcilovic | Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro[END_REF].

In addition to activation by phosphorylation, AMPK is allosterically activated by AMP [START_REF] Hardie | AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge[END_REF]. Several lines of evidence suggest that the γ subunit is involved in the allosteric activation. The γ subunit contains four CBS (cystathionine-β-synthase) domains [START_REF] Bateman | The structure of a domain common to archaebacteria and the homocystinuria disease protein[END_REF]. In human γ2 naturally occurring mutations in these domains cause cardiac hypertrophy due to massive glycogen accumulation, together with Wolff-Parkinson-White syndrome, a preexcitation disorder [START_REF] Blair | Mutations in the γ2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis[END_REF][START_REF] Gollob | Identification of a gene responsible for familial Wolff-Parkinson-White syndrome[END_REF][START_REF] Arad | Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy[END_REF]. Biochemical studies demonstrate that some of these mutations interfere with AMP-activation of AMPK [START_REF] Daniel | Functional analysis of mutations in the gamma2 Subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White Syndrome[END_REF][START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]. Furthermore, the CBS domains have been shown to bind AMP in in vitro binding assays [START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]. In contrast to AMPK, allosteric activation of SNF1 by AMP has not been demonstrated [START_REF] Woods | Yeast Snf1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo[END_REF]. As well as allosteric activation, AMP has been proposed to play a role in the phosphorylation of AMPK. Originally, three separate mechanisms were proposed whereby AMP would promote phosphorylation of AMPK. The first mechanism was direct activation of the upstream kinases by AMP [START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF].

However, the evidence supporting this mechanism was based on results obtained using partially purified preparations of the upstream kinase from rat liver, which was subsequently shown to be LKB1. More recent studies using highly purified, recombinant preparations of LKB1 reveal that AMP does not directly activate LKB1 [START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF]. Similarly, CaMKKβ is not directly activated by AMP [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF]. Two further mechanisms for promotion of phosphorylation 4 by AMP were suggested to be substrate mediated. Binding of AMP to AMPK was proposed to render the kinase a better substrate for phosphorylation by upstream kinases [START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF], whilst making it a worse substrate for dephosphorylation by protein phosphatases [START_REF] Davies | 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase -studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2A(C)[END_REF]. Although these mechanisms provide an attractive model for AMP activation of AMPK, there is relatively little direct evidence supporting them, and for these studies partially purified preparations of AMPK and the upstream kinases were used. Moreover, recent studies have reported that AMP does not promote phosphorylation of AMPK by CaMKKβ [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF]. Since both CaMKKβ and LKB1 activate AMPK by phosphorylating the same residue within AMPK it is difficult to envisage a mechanism that would account for a substrate-mediated effect of AMP that is specific for LKB1. While this manuscript was in preparation, Suter et al. reported that AMP did not promote phosphorylation of AMPK by a recombinant preparation of LKB1 [START_REF] Suter | Dissecting the role of AMP for allosteric stimulation. activation and deactivation of AMP-activated protein kinase[END_REF]. In view of the apparent discrepancy between earlier studies and more recent findings concerning the effect of AMP on phosphorylation, we decided to revisit the mechanisms for activation of AMPK by AMP using highly purified, recombinant proteins. We report here that AMP allosterically activates AMPK and inhibits dephosphorylation of T172. Both these effects involve the γ subunit. However, AMP has no effect on phosphorylation of AMPK by either LKB1 or CaMKKβ. Instead, we present evidence suggesting that previous results indicating that AMP stimulates phosphorylation of AMPK by LKB1 may have been confounded by the presence of endogenous protein phosphatase 2Cα (PP2Cα) in the preparations of the kinases used in the earlier studies.

MATERIALS AND METHODS

Materials

BL21-Codon-Plus (DE3)-RIL competent cells were obtained from Novagen. Constructs for bacterial expression of the SNF1 complex (Snf1, Snf4 and Gal83) were a kind gift from Marian Carlson (Columbia University).

Preparation of recombinant proteins

Recombinant AMPK α1β1γ1 and α2β1γ1 complexes [START_REF] Neumann | Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli[END_REF], CaMKKβ [START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF] and PP2Cα [START_REF] Marley | Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2C alpha[END_REF] were expressed in bacteria and purified as described previously. cDNAs encoding Snf1 and Snf4 were cloned into multiple cloning site 1 (SacI/HindIII) and 2 (KpnI/KpnI), respectively, of the pRSF DUET-1 vector (Invitrogen). This allowed for expression of Snf1 with an N-terminal hexa-histidine tag. cDNA coding for Gal83 was cloned into the pET DUET-1 vector (Invitrogen) at the Bg1II/KpnI sites. These constructs were cotransformed into E.coli (BL21-Codon-Plus (DE3)-RIL) and colonies were selected by growth on medium containing the appropriate antibiotic. The SNF1 complex was expressed in E.coli following induction with 1 mM isopropyl-β-thiogalactopyranoside for 4 h at 25 o C. Cells were sonicated and insoluble material removed by centrifugation. The supernatant was sterile-filtered and SNF1 complex was purified on a HisTrap HP column using an ÄKTA TM purifier (Amersham Biosciences). Following nickel-Sepharose chromatography, the SNF1 complex was further purified by gel filtration chromatography on a HiLoad 16/60 Superdex 200 column (Amersham Biosciences).

Fractions were analysed by SDS-PAGE and those containing the SNF1 complex were pooled and concentrated using a Vivaspin column (Vivascience). Authenticity of the SNF1 subunits was confirmed by mass spectrometry of the proteins resolved by SDS-PAGE and following digestion with trypsin (data not shown).

Active LKB1 complex was obtained by co-expression of His-LKB1, STRADα and GST-MO25α using a baculovirus-insect cell expression system (Bac-Bac Baculovirus Expression System, Invitrogen). The LKB1 complex was expressed in Sf9

Site-directed mutagenesis

Point mutations (R69Q, H150R and R298G) were introduced into the γ1 subunit using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The introduction of the mutations was verified by DNA sequencing.

AMPK/SNF1 assays

Recombinant AMPK or SNF1 was incubated in the presence or absence of CaMKKβ or LKB1 with 200 µM ATP, 2.5 mM MgCl 2 and 1 mM dithiothreitol for 20 min at 37 o C, and in the presence or absence of 150 µM AMP, as stated in the figure legends. In some cases, an aliquot of this reaction was taken for Western blot analysis. A separate aliquot was removed and diluted in 50 mM HEPES (pH 7.4) and AMPK or SNF1 activity was determined by phosphorylation of the SAMS peptide [START_REF] Davies | Tissue distribution of AMPactivated protein kinase, and lack of activation by cyclic AMP-dependent protein kinase, studied using a specific and sensitive peptide assay[END_REF] in the presence or absence of 150 µM AMP, as stated in the figure legends. Results shown are presented as specific activity (nmol/min/mg).

Western blot analysis

T172 (AMPK) and T210 (SNF1) phosphorylation was determined using rabbit anti-pT172 antibody (Cell Signaling). Total AMPK was detected using sheep anti-α1 or anti-α2 antibodies [START_REF] Woods | The α1 and α2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro[END_REF]. Total Snf1 was detected using mouse anti-His antibody (Abcam). PP2C was detected using mouse anti-PP2Cα antibody (Acris Antibodies). Primary antibodies were detected using LI-COR IRDye ® Infrared Dye secondary antibodies and visualised using an Odyssey Infrared Imager (LI-COR Biotechnology). Quantification of results was performed using Odyssey software and expressed as a ratio of the signal obtained with the phospho-specific antibody relative to the appropriate total antibody.

Dephosphorylation of AMPK and SNF1

Recombinant AMPK or SNF1 was phosphorylated by CaMKKβ or LKB1, as described above. An aliquot of the phosphorylated AMPK or SNF1 was incubated in 50 mM HEPES, pH 7.4, 2.

Rat liver enzymes

Rat liver AMPK was purified up to the DEAE-Sepharose step [START_REF] Carling | Purification and characterisation of the AMP-activated protein kinase[END_REF] and rat liver LKB1 up to the Q-Sepharose step [START_REF] Hawley | Characterisation of the AMP-activated protein kinase kinase from rat liver and identification of threonine-172 as the major site at which it phosphorylates AMP-activated protein kinase[END_REF]. Rat liver AMPK was dialysed into 50 mM HEPES, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 mM benzamidine, 0.1 mM phenylmethane sulphonyl fluoride, 4 µg/ml trypsin inhibitor in order to remove the protein phosphatase inhibitors present in the purification buffer. Following dialysis, AMPK was incubated in the presence or absence of 2.5 mM MgCl 2 , and the presence or absence of 50 mM NaF, plus or minus 150 µM AMP for 10 mins at 37 o C. Aliquots were removed for Western blot analysis or for AMPK activity determination using the SAMS peptide assay, measured in the presence of a final concentration of 150 µM AMP. In some cases, AMPK was incubated for 10 min at 37 o C in the presence of 200 µM ATP, 2.5 mM MgCl 2 and the presence or absence of LKB1 (16 ng), 50 mM NaF or 150 µM AMP (as stated in the figure legend). Aliquots of these reactions were removed for AMPK activity determination (measured in the presence of a final concentration of 150 µM AMP).

Statistical analysis

Results are expressed as mean values ±SEM. Statistical analysis was carried out using a two-tailed, unpaired Student's t test.

RESULTS

Effect of γ subunit mutations on allosteric activation by AMP

Two previous studies reported that naturally occurring mutations in the γ2 subunit of AMPK interfere with AMP-binding and allosteric activation of the kinase [START_REF] Daniel | Functional analysis of mutations in the gamma2 Subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White Syndrome[END_REF][START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]. The residues mutated in γ2 are conserved in all mammalian γ isoforms suggesting that they may have functionally important roles. In order to investigate more thoroughly the role of these residues in regulation of AMPK by AMP we expressed AMPK complexes in E. coli using a tricistronic expression system [START_REF] Neumann | Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli[END_REF]. In this study, we have used γ1-containing complexes because, despite extensive efforts, we have been unable to obtain purified preparations of recombinant AMPK complexes containing the γ2 isoform (data not shown). Wild type AMPK complexes (α1β1γ1 or α2β1γ1) or complexes harbouring substitution of R69Q (equivalent to R302Q in human γ2), H150R (H383R) and R298G (R531G) within the γ1 subunit, were expressed and purified by nickel-Sepharose chromatography. As can be seen in Figs. 1A and2A, no difference in expression of the various complexes was detected. As previously reported [START_REF] Neumann | Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli[END_REF], the recombinant AMPK was isolated in an inactive form, but could be activated by phosphorylation using recombinant preparations of the upstream kinases, CaMKKβ or LKB1. Following phosphorylation by CaMKKβ, AMP activated the wild-type AMPK complexes 2-fold and this activation was reduced in complexes harbouring mutations in the γ subunit (Figs. 1B, C and2B, C). The R298G mutation had the most significant effect on AMP-activation, completely abolishing activation of the α2 complex. Mutations in the γ1 subunit did not have a significant effect on the degree of phosphorylation of T172 by CaMKKβ or LKB1, nor on the specific activity of AMPK measured in the absence of AMP (Figs. 1 and2, and data not shown).

AMP does not stimulate phosphorylation of AMPK

Previous studies reported that in addition to allosteric activation, AMP also promotes the phosphorylation of AMPK by CaMKK and LKB1 [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF][START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF][START_REF] Salt | AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform[END_REF]. In the CaMKK studies, partially purified preparations from pig brain were used [START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF][START_REF] Salt | AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform[END_REF]. In subsequent studies using recombinant CaMKKα and CaMKKβ, no effect of AMP was observed [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF]. Taken together these results suggest that the effect of AMP on phosphorylation was mediated by a contaminating factor present in the pig brain preparations of CaMKK. In light of these Copyright 2006 Biochemical Society findings we decided to reinvestigate the effect of AMP on phosphorylation of AMPK by LKB1 using recombinant proteins. Wild-type AMPK complexes were activated following phosphorylation by LKB1, and under the conditions used activation was almost linear with the amount of LKB1 added (Fig. 3A and4A). Note that because AMP is present in one of the activation reactions with LKB1, we added AMP to the SAMS phosphorylation so that the final concentration of AMP is 150 µM in this assay, irrespective of whether AMP was present or absent in the LKB1 incubation. Both α1and α2-complexes were activated to similar degrees and activation correlated closely with the extent of phosphorylation of T172 (Fig. 3B and4B). Using this highly purified, reconstituted system, we did not detect any effect of AMP on phosphorylation of T172 or on activation of AMPK for either α1or α2complexes. It was reported recently that AMP is generated in the AMPK assay using the SAMS peptide as a substrate [START_REF] Suter | Dissecting the role of AMP for allosteric stimulation. activation and deactivation of AMP-activated protein kinase[END_REF]. However, at the end of the phosphorylation reaction with LKB1 and AMPK we were unable to detect AMP in the assay mixture (in the absence of added AMP), as judged by ion-exchange chromatography (data not shown).

Effect of γ mutations on AMP inhibition of dephosphorylation of AMPK

Wild-type AMPK or AMPK harbouring the R298G mutation in γ1 was phosphorylated by CaMKKβ and subsequently used as a substrate for dephosphorylation by recombinant PP2Cα. The phosphorylation state of T172 was assessed by Western blotting using anti-pT172 specific antibodies and quantified relative to the level of total α expression using an infra-red scanner. As seen in Fig 5, incubation of AMPK with PP2Cα alone led to marked dephosphorylation of the α subunit. Inclusion of AMP in the incubation with PP2Cα significantly reduced the dephosphorylation of T172 for the wild-type α1and α2-containing complexes (Fig. 5). In contrast, AMP had no significant effect on the dephosphorylation of the R298G complexes. The inhibition of dephosphorylation by AMP for the R69Q and H150R complexes was significantly reduced, though to a lesser extent than seen with the R298G complex (data not shown). Similar results were obtained using AMPK phosphorylated by LKB1. These results are consistent with a previous study that reported that AMP inhibits dephosphorylation of AMPK partially purified from rat liver [START_REF] Davies | 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase -studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2A(C)[END_REF]. Our results extend the previous findings by demonstrating that the effect of AMP on

AMP has no effect on yeast SNF1 kinase

In notable contrast to the mammalian enzyme the yeast counterpart of AMPK, SNF1, has been reported not to be allosterically activated by AMP [START_REF] Woods | Yeast Snf1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo[END_REF][START_REF] Mitchelhill | Mammalian AMP-activated Protein Kinase Shares Structural and Functional Homology with the Catalytic Domain of Yeast Snf1 Protein Kinase[END_REF], but the effect of AMP on dephosphorylation of SNF1 has not been reported. In order to investigate this, we coexpressed Snf1, Snf4 and Gal83 in E. coli. The SNF1 complex was purified by nickel-Sepharose chromatography followed by gel-filtration to near homogeneity (Fig. 6A). SNF1 was virtually inactive following purification, but could be activated by phosphorylation with CaMKKβ in vitro. Using radiolabelled ATP phosphorylation of both the Snf1 and Gal83 subunits was detected. The amino acid sequence surrounding threonine 210 (T210) in Snf1, the equivalent site to T172 in AMPK, is highly conserved between yeast and mammals, and the anti-pT172 antibody cross-reacted with phosphorylated Snf1 on Western blots (Fig. 6B).

We were therefore able to measure the dephosphorylation of T210 by PP2Cα using the anti-pT172 specific antibodies and quantify this relative to the level of total Snf1 expression using an anti-His antibody. The results shown in Fig. 6C demonstrate that AMP does not inhibit dephosphorylation of T210 in Snf1.

Partially purified AMPK and LKB1 contain endogenous PP2Cα

Our results using highly purified, recombinant preparations of AMPK, CaMKKβ, LKB1 and PP2Cα reveal that AMP allosterically activates AMPK and inhibits the dephosphorylation of AMPK, but has no effect on phosphorylation by upstream kinases. The latter finding is at odds with several previous studies using partially purified preparations of AMPK and LKB1. One possibility that could explain this apparent discrepancy is that these preparations contain a factor that mediates the effect of AMP on phosphorylation. Based on our current results we reasoned that this factor could in fact be a protein phosphatase. To test this, we used a partially purified AMPK preparation in an activation assay with recombinant LKB1. AMPK was activated approximately 2-fold by LKB1 and AMP increased activation a further 2-fold (Fig. 7A). However, if NaF, a protein phosphatase inhibitor, was included in the reactions, AMPK activity was increased almost 6-fold by LKB1, and was not further In order to determine directly the presence of a protein phosphatase in a partially purified preparation of rat liver AMPK (purified up to the first ion-exchange step [START_REF] Carling | Purification and characterisation of the AMP-activated protein kinase[END_REF]) we adopted a shotgun mass spectrometry approach. The results (not shown) revealed the presence of PP2Cα, and this was confirmed by Western blot analysis (Fig. 7B). To investigate whether PP2Cα activity is present in the AMPK preparation we incubated AMPK in the presence or absence MgCl 2 , an essential co-factor for PP2Cα [START_REF] Mcgowan | Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+-dependent enzyme[END_REF], and determined AMPK activity and T172 phosphorylation (Fig. 7C,D). AMPK activity and T172 phosphorylation were significantly reduced only in the presence of MgCl 2 and these decreases were totally blocked by the inclusion of 50 mM NaF. Consistent with our studies using recombinant AMPK and PP2Cα, AMP significantly inhibited, but did not abolish, these effects. PP2Cα was undetectable in more highly purified preparations of AMPK (up to the gel-filtration step [START_REF] Carling | Purification and characterisation of the AMP-activated protein kinase[END_REF]), or following immunoprecipitation. Using these preparations, AMPK activity was not affected following incubation with MgCl 2 . Furthermore, we did not detect an effect of AMP on activation of immunoprecipitated AMPK by recombinant LKB1 (data not shown). However, AMP-stimulation of T172 phosphorylation has been reported using more highly purified preparations of AMPK [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF][START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF]. In these studies, LKB1 was purified from rat liver up to the Q-Sepharose step [START_REF] Hawley | Characterisation of the AMP-activated protein kinase kinase from rat liver and identification of threonine-172 as the major site at which it phosphorylates AMP-activated protein kinase[END_REF], raising the possibility that PP2Cα could be present in the preparations. We found that PP2Cα was readily detectable by Western blotting in rat liver LKB1 purified to the same stage (Fig. 7E).

DISCUSSION

In this study we investigated the mechanisms by which AMP leads to activation of AMPK. We took advantage of the use of highly purified, recombinant preparations of the components of the AMPK cascade. Wild-type AMPK complexes (α1β1γ1 and α2β1γ1) expressed in bacteria were activated by phosphorylation using either CaMKKβ or LKB1, as has been reported previously [START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF][START_REF] Woods | Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells[END_REF][START_REF] Neumann | Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli[END_REF]. AMP allosterically activated both α1and α2containing complexes by approximately 2-fold. The degree of activation of recombinant α1β1γ1 is slightly greater than that reported previously for α1γ1 complexes isolated by sequential immunoprecipitation from rat brain (2.1-fold vs 1.7-fold), whereas activation of recombinant α2β1γ1 is slightly lower than that reported for α2γ1 complexes from rat brain (2.1-fold vs 2.8-fold) [START_REF] Cheung | Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding[END_REF]. A complication with the measurements using native rat brain AMPK is that these complexes would contain a mixture of β1 and β2 isoforms, and this might account for the minor differences in AMP-stimulation observed between the two studies.

The γ subunit isoforms contain 4 copies of a CBS (cystathionine-β-synthase) domain and previous studies have shown that these domains are involved in AMP-binding and subsequent allosteric activation of AMPK [START_REF] Daniel | Functional analysis of mutations in the gamma2 Subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White Syndrome[END_REF][START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Hamilton | An activating mutation in the g1 subunit of the AMP-activated protein kinase[END_REF]. Part of the evidence implicating the CBS domains in regulation of AMPK comes from the identification of naturally occurring mutations in γ2 that cause severe cardiac defects in humans [START_REF] Blair | Mutations in the γ2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis[END_REF][START_REF] Gollob | Identification of a gene responsible for familial Wolff-Parkinson-White syndrome[END_REF][START_REF] Arad | Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy[END_REF], and a naturally occurring mutation in γ3 in pig that leads to glycogen accumulation in skeletal muscle [START_REF] Milan | A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle[END_REF].

Most of these mutations lie within the CBS domains and interfere with the normal activation of AMPK by AMP [START_REF] Daniel | Functional analysis of mutations in the gamma2 Subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White Syndrome[END_REF][START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF]. For reasons that are unclear, we have been unable to express γ2or γ3-containing complexes in bacteria and so we introduced substitutions in the γ1 subunit to mimic three of the equivalent naturally occurring mutations found in γ2, including the equivalent mutation in pig γ3. Complexes containing the mutant γ1 subunits were expressed in bacteria and were activated by CaMKKβ or LKB1, similar to wild-type AMPK.

Consistent with previous studies, AMP-activation was reduced in the γ1-mutant complexes, with the R298G mutation having the greatest effect.

Davies et al. reported previously that AMP inhibits dephosphorylation of AMPK [START_REF] Davies | 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase -studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2A(C)[END_REF]. We confirmed that AMP has a significant inhibitory effect on dephosphorylation of recombinant AMPK by PP2Cα. Whilst our manuscript was in preparation, Suter et al. Copyright 2006 Biochemical Society published results using recombinant AMPK in which they also demonstrated AMP inhibition of dephosphorylation [START_REF] Suter | Dissecting the role of AMP for allosteric stimulation. activation and deactivation of AMP-activated protein kinase[END_REF]. Importantly, we now show that a mutation in CBS4 of AMPK (R298G) completely abolishes the effect of AMP on dephosphorylation. Furthermore, the R298G mutation almost completely abolishes allosteric activation of AMPK, suggesting that the same mechanism may account for both AMP effects. SNF1, the yeast counterpart of AMPK, is activated by phosphorylation of T210, the equivalent residue to T172 in AMPKα. Although SNF1 does not appear to be directly activated by AMP [START_REF] Woods | Yeast Snf1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo[END_REF][START_REF] Mitchelhill | Mammalian AMP-activated Protein Kinase Shares Structural and Functional Homology with the Catalytic Domain of Yeast Snf1 Protein Kinase[END_REF], there are no reports as to whether AMP has an effect on its dephosphorylation. We were able to purify a SNF1 complex following co-expression of Snf1, Snf4 and Gal83 subunits in bacteria. As with AMPK, SNF1 was isolated in an inactive state, but could be activated by phosphorylation in vitro using CaMKKβ or LKB1 (data not shown). Consistent with the previous studies, we did not detect any allosteric activation of recombinant SNF1 by AMP. In addition, we were unable to detect any effect of AMP on dephosphorylation of T210 by PP2Cα. In yeast, SNF1 is activated following growth under glucose limiting conditions [START_REF] Carlson | Glucose repression in yeast[END_REF]. The signal transduction pathway linking glucose limitation to SNF1 activation is unclear, but our results suggest that AMP is unlikely to be directly involved. However, the availability of a reconstituted system for SNF1 activation would allow potential activators, such as glucose metabolites, to be investigated.

Previous studies reported that phosphorylation of AMPK by preparations of an AMPK kinase (AMPKK) from rat liver was stimulated by AMP [START_REF] Hawley | 5'-AMP Activates the AMP-activated protein kinase cascade and Ca 2+ /calmodulin activates the calmodulin-dependent protein kinase-I cascade, via 3 independent mechanisms[END_REF]. Subsequent work has revealed that the identity of AMPKK in these preparations is LKB1 [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF]. Using recombinant preparations of AMPK and LKB1, however, we have been unable to detect any stimulation of T172 phosphorylation by AMP under any conditions. Recently, Suter et al. published results using recombinant AMPK in which they also failed to detect an effect of AMP on T172 phosphorylation by LKB1 or CaMKKβ [START_REF] Suter | Dissecting the role of AMP for allosteric stimulation. activation and deactivation of AMP-activated protein kinase[END_REF]. However, the authors were unable to account for the apparent discrepancy between their results and previous studies. Here we provide a plausible explanation for this inconsistency. We reasoned that the partially purified preparations of AMPK and/or LKB1 used in previous studies examining AMP-stimulation could contain an additional factor that mediates the effect of AMP on T172 phosphorylation.

In an effort to investigate this further, we began by adopting a shotgun mass spectrometry approach to determine proteins present in a partially purified preparation of rat Copyright 2006 Biochemical Society liver AMPK (purified up to the first ion-exchange step [START_REF] Carling | Purification and characterisation of the AMP-activated protein kinase[END_REF]). The results (not shown) revealed the presence of PP2Cα, and this was confirmed by Western blot analysis. We present strong evidence that the AMP effect on T172 phosphorylation is due to the presence of PP2Cα since we only observe AMP-stimulation using preparations of AMPK that contain PP2Cα and the effect is abolished by NaF. Based on these findings we suggest that AMPstimulation of T172 phosphorylation is due to inhibition of PP2Cα present in the preparations of AMPK and/or LKB1 and CaMKK. In support of this hypothesis, we showed that PP2Cα is present in a preparation of rat liver LKB1. Whilst we cannot rule out other possibilities, this explanation would account for the differences between our studies using defined, purified preparations of recombinant proteins and those studies using less welldefined preparations. It is interesting to note that all the studies reporting AMP-stimulation of T172 phosphorylation use okadaic acid to inhibit dephosphorylation, but do not include NaF. Since PP2Cα is okadaic acid insensitive [START_REF] Bialojan | Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics[END_REF], it would remain active under the conditions of the assay (since PP2Cα would be activated by the Mg 2+ present in the kinase reaction).

Overall, our results lead us to propose a revised model for activation of AMPK. The phosphorylation state of T172 depends on the relative rates of phosphorylation, catalysed by CaMKKβ and LKB1, and dephosphorylation, catalysed by protein phosphatases (Fig. 8). The identity of the protein phosphatase(s) acting on AMPK in vivo is unknown, however, the effect of AMP on dephosphorylation is substrate mediated [START_REF] Davies | 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase -studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2A(C)[END_REF] and so AMP would be predicted to inhibit all phosphatases acting on AMPK. Activation of AMPK occurs either by increased phosphorylation or by decreased dephosphorylation. LKB1 appears to be constitutively active and furthermore its activity is not changed by stimuli that activate AMPK [START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF][START_REF] Sakamoto | Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR[END_REF]. According to our model, increased T172 phosphorylation by LKB1 would occur in response to decreased dephosphorylation following a rise in AMP. In parallel, AMP would allosterically activate AMPK (Fig. 8A). Dephosphorylation and inactivation of AMPK would occur when the concentration of AMP returned to basal levels. Unlike LKB1, the activity of CaMKKβ is subject to regulation within the cell, and is increased in response to signals that raise intracellular Ca 2+ [START_REF] Tokumitsu | Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms[END_REF][START_REF] Anderson | Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta[END_REF]. Therefore, signals that increase Ca 2+ activate AMPK as a consequence of increased T172 phosphorylation via increased CaMKKβ activity. Under these conditions there is no requirement for increased AMP levels (Fig. 8B). Copyright 2006 Biochemical Society mediated signalling without detectable changes in AMP [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Stahmann | Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta[END_REF][START_REF] Tamas | Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes[END_REF]. A further prediction of our model is that AMPK would be rapidly dephosphorylated following a fall in intracellular Ca 2+ , and this may account for the transient nature of AMPK activation observed in CaMKKβ-mediated responses [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Stahmann | Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta[END_REF][START_REF] Tamas | Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes[END_REF]. It should be noted that the AMP-and Ca 2+signalling pathways for activation of AMPK predicted by our model are not mutually exclusive. It is possible that under some circumstances, where AMP and Ca 2+ rise in concert, both pathways could operate. Under these conditions, both CaMKKβ and LKB1 would contribute to AMPK activation.

Consistent with this model, recent studies have reported activation of AMPK by CaMKKβ-

Although our results were obtained with γ1-containing AMPK complexes we speculate that the same mechanisms will apply to γ2and γ3-containing complexes. Previous studies have shown that mutations within γ2 and γ3 interfere with allosteric activation of AMPK measured in vitro [START_REF] Daniel | Functional analysis of mutations in the gamma2 Subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White Syndrome[END_REF][START_REF] Scott | CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[END_REF][START_REF] Barnes | The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle[END_REF] and so it seems likely that these mutations will also interfere with the effect of AMP on dephosphorylation. Two studies have reported that γ2 mutations decrease AMPK activity in transgenic animal models [START_REF] Sidhu | Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff-Parkinson-White syndrome[END_REF][START_REF] Davies | Characterization of the role of gamma2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and Wolff-Parkinson-White syndrome[END_REF], whereas another study reported increased activity [START_REF] Arad | Transgenic Mice Overexpressing Mutant PRKAG2 Define the Cause of Wolff-Parkinson-White Syndrome in Glycogen Storage Cardiomyopathy[END_REF]. In a study in which γ3-containing AMPK complexes were over-expressed in COS cells, increased activity was observed in mutant γ3 relative to wild-type [START_REF] Barnes | The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle[END_REF]. Despite our understanding of the regulation of AMPK by AMP in vitro, elucidating the complex regulation of AMPK in cells and in vivo, and determining the effect of mutations within the γ subunits, is far from complete.

In conclusion, our results demonstrate that AMP activates AMPK via a direct allosteric mechanism and indirectly, by inhibiting dephosphorylation of T172. These effects may be mediated by binding of AMP to the same site within the γ subunit, since both effects are abolished by the same mutation within the γ subunit. We provide evidence that suggests that previous studies reporting activation of T172 phosphorylation by AMP may have been mediated by PP2Cα present in the partially purified preparations of the kinases. Based on our results we propose a model for regulation of AMPK that accounts for its activation in response to either Ca 2+ -signalling or energy depletion, mediated by an increase in AMP. The same set of experiments as described for Figure 1 were carried out using α2 complexes. (A) AMP-dependent activation: Under conditions that lead to an increase in AMP, dephosphorylation of AMPK is inhibited (mechanism 1). The identity of the protein phosphatase (PP) responsible for dephosphorylation of AMPK in vivo is unknown. Since LKB1 is constitutively active, inhibition of the dephosphorylation reaction leads to an increase in T172 phosphorylation and activation of AMPK. In addition to increasing T172 phosphorylation, AMP allosterically activates AMPK (mechanism 2). (B) Ca 2+ -dependent activation: Signals that increase Ca 2+ activate CaMKKβ, increasing T172 phosphorylation and activation of AMPK and this can occur without an increase in AMP. However, it is possible that in some situations both Ca 2+ and AMP may increase in parallel, and under these conditions AMP will allosterically activate AMPK and inhibit dephosphorylation (denoted by the dashed lines).

  cells and was purified by chromatography on glutathione-Sepharose. Recombinant protein was stored in 50 mM Hepes, pH 7.4, 200 mM NaCl, 10% glycerol and 1 mM Tris (2-carboxyethyl) phosphine hydrochloride at -80 o C. Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society

  5 mM MgCl 2 , in the presence or absence of recombinant PP2Cα (26 ng) and the presence or absence of 150 µM AMP for 20 min at 37 o C. The reaction was terminated by the addition of gel-loading buffer. Samples were resolved by SDS-PAGE and subjected to Western blot analysis. Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society Sanders et al. Mechanism of AMPK activation by AMP 10 dephosphorylation involves residues that are also involved in the allosteric regulation of AMPK.

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520Copyright 2006 Biochemical Society activated by the addition of AMP, i.e. AMP and NaF have similar and non-additive effects on AMPK phosphorylation. Rather than showing an effect of AMP to promote phosphorylation, these data are most consistent with the hypothesis that AMP acts to inhibit the dephosphorylation of AMPK with the overall effect being an increase in AMPK activity.

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520

Biochemical

  Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520

Figure 1 .

 1 Figure 1. Effect of mutations within γ subunit on the allosteric activation of α1β1γ1 by AMP. Wild-type (WT) AMPK complex (α1β1γ1) or complexes harbouring point mutations in the γ subunit (R69Q, H150R or R298G) were expressed in E. coli and purified by nickel-Sepharose chromatography. (A) Proteins (~1 µg) were resolved by SDS-PAGE and detected by staining with Coomassie. (B, C) Following phosphorylation by CaMKKβ, AMPK activity of the complexes was determined using the SAMS peptide assay in the presence or absence of 150 µM AMP. Results are the average ± S.E.M. for 4 independent experiments and are shown as the specific activity of the kinase (nmole phosphate incorporated/min/mg complex) in (B) and the fold-stimulation in (C). (D) Western blot analysis of AMPK complexes probed with an antibody against either phospho-T172 (pT172) or α1. A representative blot is shown in the upper panel and a graph showing relative quantitation of pT172:total α1 from 4 independent experiments (average ± S.E.M.) is shown below. Wild-type AMPK that had not been incubated with CaMKKβ is included as a negative control.

Figure 2 .

 2 Figure 2. Effect of mutations within γ subunit on the allosteric activation of α2β1γ1 by AMP.

Figure 3 .

 3 Figure 3. Effect of AMP on the phosphorylation of α1β1γ1 by LKB1. (A) Wild-type α1β1γ1 was incubated with MgATP and varying amounts of LKB1 in the presence (closed symbols)or absence (open symbols) of 150 µM AMP for 10 min at 37° C. At the end of the incubation an aliquot was removed, diluted in buffer and AMPK activity determined using the SAMS assay. Note that in the SAMS assay, AMP (150 µM final concentration) is present irrespective of whether AMP was included in the initial incubation with LKB1. (B) A representative Western blot of pT172 and total α1 of aliquots of the reactions described above is shown. A graph of the relative quantitation of pT172:total α1 from 3 independent experiments (average ± S.E.M.) is also shown.

Figure 4 .

 4 Figure 4. Effect of AMP on the phosphorylation of α2β1γ1 by LKB1. The same set of experiments as described for Figure 3 were carried out using α2 complexes.

Figure 5 .

 5 Figure 5. Effect of AMP on the dephosphorylation of recombinant AMPK by PP2C. Following phosphorylation of recombinant AMPK complexes (containing either wild type or R298G γ1) by CaMKKβ, aliquots were removed and incubated in the presence or absence of PP2C (26 ng) and presence or absence of AMP (150 µM) for 20 min at 37°C. At the end of the incubation the reaction mixture was resolved by SDS-PAGE and analysed by Western blotting using anti-pT172 and anti-α1 (A) or anti-pT172 and anti-α2 (B) antibodies. In each case a representative blot is shown. A graph of pT172 phosphorylation relative to a control (absence of PP2C and AMP) is shown (average ± S.E.M. of 4 independent experiments). Significant differences in T172 phosphorylation are denoted by * (p<0.05). Non-significant results are denoted NS.

Figure 6 .

 6 Figure 6. Effect of AMP on recombinant SNF1 complex. SNF1 (Snf1, Snf4, Gal83) was expressed in E. coli and purified by nickel-Sepharose, followed by gel-filtration chromatography. (A) Purified SNF1 was incubated with 32 P-ATP in the presence or absence of CaMKKβ and resolved by SDS-PAGE. Protein was visualised by staining with Coomassie (upper panel) and radiolabelled products detected by autoradiography (lower panel). (B) SNF1 activity was determined by the SAMS peptide assay in the presence or absence of 150 µM AMP. Activities are plotted as nmole phosphate incorporated per min per mg SNF1 and are the average ± S.E.M from 3 independent experiments. Also shown is a representative blot of the reaction mixture probed with anti-pT172 (to detect pT210 in Snf1) or anti-His antibody (to detect total Snf1). (C) SNF1 phosphorylated by CaMKKβ was incubated in the presence or absence of PP2C (26 ng) and the presence or absence of 150 µM AMP for 20 min at 37°C. At the end of the incubation proteins were analysed by Western blotting using either anti-pT172 or anti-His antibody as described for Figure 5. Results shown are the average ± S.E.M. from 4 independent experiments.

Figure 7 .

 7 Figure 7. Inactivation of rat liver AMPK by endogenous protein phosphatase activity. (A)Following dialysis into buffer lacking protein phosphatase inhibitors, rat liver AMPK was incubated for 10 min at 37°C in the presence of MgATP and the presence or absence of LKB1 (16 ng), AMP (150 µM) and/or NaF (50 mM). Following the incubation, AMPK

Figure 8 .

 8 Figure 8. Model for the regulation of AMPK.

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society

ACKNOWLEDGEMENTS

Biochemical Journal Immediate Publication. Published on 6 Dec 2006 as manuscript BJ20061520 Copyright 2006 Biochemical Society