It has been observed clinically that the Z-effect is a potential cause of failure of an intramedullary nail with two cephalic screws. It describes the migration behavior of the cephalic screws in the femoral head. The primary objective was to examine different cephalic screw configurations and test the load distribution between them as a function of their relative placement and their relative movement in the nail. It has been hypothesized that different cephalic screw positions may have an influence on the stress in the implant and bone and therefore on implant failures, such as the Z-effect.To quantify the load distribution of a dual cephalic screw intramedullary femoral nail , a finite element model of the femur, focusing on the loading of the cephalic screws, was prepared. Four different screw lengths (90–105 mm) were examined. The investigation considered the stresses and strains in the bone and implant as well as the relative movement of the screws.If the inferior cephalic screw had a shorter length, then the superior one and the femoral nail had to bear higher loads. In that case, the “equivalent von Mises stress” increased up to 10 % at the superior cephalic screw and up to 5 % at the femoral nail. The analysis of the relative movement showed that sliding of the inferior cephalic screw occurred in the nail. The total movement ranged from 0.47 to 0.73 mm for the different screw configurations.The stresses were distributed more equally between the two cephalic screws in the bone and the implant if a longer inferior screw was used. The stresses in the bone and implant were reduced with a longer inferior cephalic screw. Therefore, a configuration using a longer inferior cephalic screw is preferable for trochanteric fracture fixation with a dual cephalic screw intramedullary device. The reported number of proximal femoral fractures has increased steadily and due to aging populations, a further increase can be expected , 2. The Intramedullary devices for the fixation of proximal femur fractures are available with single, dual, and two integrated lag screws. Clinical experience with single and integrated screw design is most favorable for intertrochanteric fractures and subtrochanteric fractures. Dual lag screw designs may have a role for medially located trochanteric fractures or lateral fractures of the femoral neck and have demonstrated sufficient mechanical stability , 11. HowOne important factor affecting failure of implants in the femoral head is their localization within the head. For extramedullary implants, the tip to apex distance (TAD) has been identified as an important factor predicting cut-out through the femoral head. It has been shown that cut-out is more likely with an increasing value of TAD –17.Also for intramedullary implants, the location of the cephalic screw within the femoral head has been suggested as an important factor influencing the failure of implant fixation , 19. TheTherefore, the hypothesis of our study was that the cephalic screw positions in a dual cephalic screw design affect the load distribution in the implant and the surrounding bone. The specific aims of this computational analysis were to determine the local stress distributions and micro-movements under load application for different cephalic screw configurations.2 intramedullary femoral nail . The reaction loads at the support of the cephalic screws in the nail, and the maximum strain/stress on the cephalic screws as a response to the applied hip joint force and the fixation of the shaft, were both assessed for different positions of the cephalic screw tips.To quantify the load distribution, a finite element (FE) analysis of the proximal femur was prepared with emphasis on the loading of the two cephalic screws in a cephalomedullary implant for proximal femur fracture fixation. The model comprised a proximal fracture of the femur fixed with an EBAThe study was divided into two parts. In the first part, a mechanically stable configuration representing ideal repositioning of the fracture fragments was modeled. In the second part, an unstable configuration representing inadequate repositioning, reduced bone quality, and sliding of the cephalic screw was investigated.2 nail system. The implant was inserted according to the surgical manual, with no interfragmentary gap. The implant material was modeled as Titanium Alloy Ti6Al4V with material constants: Elastic Modulus E [MPa] 115,000; Poisson’s ratio μ 0.342.The FE model in this study was based on a standardized femur geometry with homogenous bone material properties for cortical and cancellous bone  which has been previously described in detail . The modThe boundary conditions of the FE model mimicked a physiological load in stance. The knee joint was simulated as a cardanic joint and the hip as a ball and socket joint. A force of 1900 N was applied at the center of femoral head, which represents the physiological load of the hip joint during walking for an 80 kg person .The model of the femur including the fracture and the intramedullary nail was created using FE software . Full models were designed with different contact types to determine which contact behavior was the most suitable. For further studies, the “simplified friction multipoint constraint contact (MPC)” model was used. This means that the threads of the cephalic screws are simplified and bonded by a multipoint constraint formulation. Thus in the stable configuration simulation, no sliding of the cephalic screws could occur in the proximal fragment of the bone. All FE analyses were conducted with ANSYS .For validation purposes, the FE model of the stable trochanteric fracture was constructed as a mechanical model using a composite adolescent-sized femur . The simulated models were validated by four mechanical tests. One test was performed as a reference on the intact surrogate femur. Three further tests used different lengths of cephalic screws .An experienced surgeon performed the surgery and created a fracture with a bone saw. The femoral head was embedded in a hemisphere with casting resin  to ensure adaption to the test machine. Thereby, the hemispherical shape represented the hip-like ball joint. The distal part of the femur was embedded to fit into a custom made cardanic joint. The mechanical tests were conducted with a servo-electric testing machine . A quasi-static axial compression test with a load ramp up to 1000 N was performed. For the validation, the experimental and simulated results were compared for axial stiffness, bone deformation, and strains at the bone and the implant.2 nail was fitted with four SGs: one posteriorly above the cephalic screw holes and one anteriorly below the screw holes. Additionally, one SG was fixed at every cephalic screw. The SGs were coupled to an amplifier . The recording and the evaluation of the data was performed with additional software . Local displacements were detected by optical markers on the bone surface and an optical tracking system  which recorded the displacements of optical markers on the bone surface with an accuracy of 0.025 mm.The strains were measured by strain gauges (SG) . The bone was fitted with four SGs. One SG was fixed superiorly and one inferiorly on the femoral neck. The other two SGs were fixed medially and laterally on the femur shaft. The EBA2 femoral nail as a function of their relative placement. Strains and stresses were examined at the bone and the implant with emphasis on the stresses at the cephalic screws and in the surrounding bone. The stresses were recorded from the straight part of the screw before the chamfer, and the relative movements of the cephalic screws within in the nail were investigated. Four different insertion lengths with a range of 90 to 105 mm were examined for each cephalic screw, so there was a total of 16 different screw configurations.The primary objective of the study was to examine different cephalic screw configurations and determine the load distribution between the two cephalic screws in the EBAThe computational model for the unstable configuration was an extended version of the validated model of the stable configuration. Compared to the stable model, it contained modification of the fracture, the bone quality, and the screw geometry.The unstable model was created with a fracture without any bone contact Fig. . The fraThe quality of the bone was changed. In the unstable model, the Young’s modulus of the cancellous bone was reduced to 80 MPa, which represented a bone with low bone quality. The unstable fracture with low bone quality represented a worst-case scenario.f 0.08 [While in the stable model a simplified screw model without threads was used, the unstable model included simplified circular threads. Additionally, the contact between the screw and the bone was changed. This allowed for the screws to slide with a resistance proportional to the frictional coefficient of f 0.08 . This coThe examined stresses are equivalent von Mises stresses. Only partially and specifically indicated normal stress was examined at the screws.The percentage error between numerical and experimental result was largest for the strain measurements (−36 % ± 27 % (mean ± SD)) and smallest for measurements of axial stiffness (−10 % ± 12 %). While strain and stiffness appeared to be underestimated by the FE analysis, local displacements were overestimated by about 25 % ± 21 %.Comparing the stress on the implant including the cephalic screws, it could be observed that across all combinations of screw lengths, the difference between the stresses in the implant was less than 3 % Fig. .Fig. 2“EThe maximum stress on nail was always observed in the area around cephalic screw holes of the intramedullary nail Fig. . The strThe largest stresses on the screws occurred around the chamfer and reached a maximum of 1168 MPa at the superior screw and 1078 MPa at the inferior screw Fig. . On averThe stress in the cortical bone of the femoral neck changed for different cephalic screw configurations. With decreasing length of the inferior cephalic screw, the stress increased, especially in the inferior cortex and also at the anterior aspect of the transition between neck and head Fig. .Fig. 4“EThe stress maxima in the cancellous bone of the proximal fragment were found in between and above the two cephalic screws. As the length of the screws decreased, the maxima shifted from the head to the neck area Fig. . AnotherThe stress distribution at the nail in the unstable configuration was similar to that of the stable fracture situation. The stress maxima were located around the bore holes and reached values of up to 1748 MPa at the nail Fig. . In the The maximum stress in the cephalic screws occurred at the posterior surfaces of both screws. With a shorter inferior screw, the superior cephalic screw had to bear higher loads. This resulted in increased tensile normal stress for the superior screw and reduced compressional normal stress for the inferior screw Fig. .Fig. 7NoTherefore, the normal stresses were the highest at the superior screw for the 105/90 configuration at 794 MPa and for the configuration with the smallest screws (90/90) at 793 MPa. For those two configurations, the stresses at the inferior cephalic screws were lowest. The normal stresses at the inferior screw were 432 MPa for the 105/90 configuration and 439 MPa for the 90/90 configuration.The stress difference between the superior and the inferior screw was smallest for configurations with longer inferior screws. The normal stress difference was 252 MPa for the configuration with the longest screws and 233 MPa for the 90/105 configuration.Similar to the stable configuration, stress in cortical bone concentrates in the inferior location of the neck to head region. The stress increases when the length of the inferior cephalic screw decreases Fig. .Fig. 8“EThe stress distribution within the cancellous bone is similar for the stable and unstable configurations. The stress maxima occurred between the two screws and above them Fig. . HoweverThe relative movement of the screws within the nail was calculated to determine possible sliding of the screws. A lateral sliding of the inferior screw was observed for all screw configurations. The largest sliding of the inferior screw was 0.7 mm. This was observed in the configuration with both screws having the smallest length (90 mm/90 mm). In contrast, the sliding of the superior screw remained below 0.03 mm for all of the configurations Fig. .Fig. 10SThe objective of this study was to analyze the effects of using different configurations of lag screw placement on the mechanical behavior of a dual lag screw trochanteric nail. Our findings indicate that by using a longer inferior lag screw, stress within the bone, the nail and the superior lag screw can be reduced and the difference in stress between the superior and inferior screws was smallest with a longer inferior screw. The length of the superior screw had minor influence on the stress in the bone and within the implant. The analysis of screw displacement within the nail demonstrated a lateral migration of the inferior screw during loading.Due to the fact that our study represents a FE study, the preferred cephalic screw configuration is a suggestion and has to be proved by further mechanical tests and clinical investigations. Computational models require simplifications of real-world situations and thus always have inherent limitations. Our FE model simulated only one type of fracture with two variations of bone density and two qualities of fracture reduction and therefore represents only a limited part of the large clinical variety of trochanteric fracture situations. The bone model used in the simulation was based on a simplified biomechanical bone model which has been previously validated . While tTwo different FE models were used in this study to analyze implant loading for different lag screw configurations. While the stable model simulated an ideal fracture reposition in a healthy bone, the unstable model simulated a worst-case scenario with imperfect fracture reposition and reduced bone quality. In the stable model, the effect of different lag screw configurations on the load distribution in the implant bone construct was rather small. This would indicate that the treatment of trochanteric fractures is not sensitive to the placement of the lag screws within the limits investigated in this study. In the unstable model, the effect of cephalic screw placement on the load distribution of the implant bone construct became more pronounced. All of the parameters assessed in the FE study were affected by lag screw placement. The most dominant effects were found for screw displacement and stress within the bone.The values of stress within the bone and the implant material can indicate potential locations of failure or fatigue. Stresses in the proximal bone fragment were largest at the inferior location of the femoral neck and reached values of up to 50 MPa. Although these stress values were still below the strength of femoral cortical bone (180–209 MPa) , the locIn our study, the lateral sliding of the inferior lag screw during loading is consistent with the description of the clinically observed Z-effect in dual lag screw implants, in which the inferior lag screw migrates laterally, and the superior lag screw migrates medially during physiologic loading. The lateral sliding was largest if the inferior screw had the shortest length. This indicates that a short insertion depth of the inferior screw would support the occurrence of the Z-effect. It has to be noted, however, that in this study the model was tested under a static load, while the Z-effect is assumed to be a phenomenon which occurs under cyclic loading . The cliIn conclusion, our results demonstrate that the configuration of cephalic screw placement in dual cephalic screw implants influences the load distribution at the implant and the bone. The size of the effect depends on the stability of the osteosynthesis and becomes more relevant in osteoporotic bone and imperfect fracture reduction. The most favorable load distribution with reduced overall stress was achieved using longer inferior cephalic screws. This could potentially decrease the likelihood of implant failure, varus collapse, and occurrence of screw migration. These biomechanical findings confirm clinical recommendations for this implant type.


Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.  •Milligram quantities of ubiquitin phosphorylated at Ser 20, 57, or 65 are purified•Twenty isomeric phosphoubiquitin dimers are assembled and purified•UBE3C chain synthesis specificity is controlled by Ser 20 ubiquitin phosphorylation•Phosphorylation of ubiquitin controls deubiquitinase activity and linkage specificity Huguenin-Dezot et al. combine genetic code expansion and enzymatic assembly to synthesize 20 isomeric phosphoubiquitin chains with distinct Ser phosphorylation and/or isopeptide linkage sites. They discover that ubiquitin phosphorylation can control E3 ligase specificity and deubiquitinase specificity. The post-translational modification of proteins with ubiquitin (Ub) modulates an expanding array of cellular processes . Ub is aThe action of the E1, E2, and E3 cascade is counteracted by deubiquitinases (DUBs) that cleave the isopeptide bond between the target protein and Ub. There are five established families of DUBs: Ub-specific proteases (USPs), Ub C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephins, and JAMMs . The intMany proteins have been identified as targets of ubiquitination. Proteins can be mono-ubiquitinated or modified with Ub polymers, in which Ub is linked via an isopeptide bond between the C terminus of one monomer and a lysine residue  or via a peptide bond to the N terminus (M1) of another monomer.Ub linkages have been identified in cells at varying abundances . BecauseUb is a target for post-translational modifications, including acetylation  and phosCUA pairs . We determined the consequences of each phosphorylation for E1 activation and E2 conjugation by 18 E2s. We assembled and purified 20 phosphorylated Ub dimers, with distinct sites of peptide linkage  and phosphorylation , using the E2/E3 combinations reported for the synthesis of unmodified Ub chains. In the process of assembling and characterizing phosphoubiquitin chains, we discovered that phosphorylation of serine 20 on Ub converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. DUB assays with phosphorylated Ub dimers revealed that (1) phosphorylation at distinct sites on Ub can control DUB-mediated cleavage of a given Ub linkage, and that (2) phosphorylation at each site in Ub can control the specificity with which DUBs cleave different Ub linkages.Here we used genetic code expansion to express and purify multi-milligram quantities of Ub bearing homogeneous phosphorylation at Ser20 (Ub [pSer20]), Ser57 (Ub [pSer57]), and Ser65 (Ub [pSer65]) from CUA pair, an approach that we previously demonstrated allows the production of homogeneous and site-specifically phosphorylated Ub and other phosphoproteins -His6 gene, where XX defines the site of serine phosphorylation in the protein, TAG is the amber codon, and His6 encodes the hexahistidine tag. Phosphoserine was directed into the protein using an evolved SepRS/tRNAproteins . Ub , which diffracted to 1.55 Å  and showCUA pair . The folCUA pair D, thoughCUA pair E.To test whether phosphorylation at serine in position 20, 57, or 65 influenced the first two steps in the ubiquitination cascade, E2-charging reactions containing an E1 (Ube1), ATP, and 18 different E2s were performed A–2R. We Next we asked whether Ub (pSer20), Ub (pSer57), and Ub (pSer65) chains linked through M1, K6, K11, K29, K33, K48, or K63 on Ub can be formed using combinations of enzymes that are known to assemble these chains with unmodified Ub . We follFor the enzymes that assemble M1-linked linear Ub chains , the extent of chain formation was not attenuated with Ub (pSer20) and Ub (pSer57) A. HoweveFor the enzymes that assemble K6 chains , using K48R mutants of Ub to avoid forming K48 chains, we observed no substantial differences in the extent of Ub chain formation with the K48R Ub species tested B. HoweveK11- and K63-linked Ub chains can be formed by Ube1 and UBE2S-UBP. The addition of AMSH removes K63 chains leaving pure K11-linked Ub. We observed no substantial effect on the extent of chain formation with Ub (pSer20), Ub (pSer57), or Ub (pSer65) C, and UbA mixture of K29- and K48-linked chains can be assembled by Ube1, UBE2D3, and UBE3C. Addition of the DUB vOTU cleaves K48 chains in this mixture, leaving pure K29 chains . We obseA mixture of K33-, K11-, and K48-linked chains can be assembled with Ube1, UBE2D1, and AREL1. To create pure K33 chains, the DUBs Cezanne-EK and OTUB1 are added to hydrolyze K11 and K48 chains, respectively, and leave pure K33 chains . The phoWe used an E1/E2 combination (Ube1 and UBE2R1) that assembles K48 chains with Ub for chain assembly with each of the phosphorylated Ubs. We found that chain assembly was attenuated for Ub (pSer65) F and S2.We used an E1/E2 combination  that assembles K63 chains with unmodified Ub for chain assembly with each of the phosphorylated Ubs. We observed a modest attenuation of chain assembly for Ub (pSer20) and Ub (pSer57) and a more dramatic attenuation of chain assembly for Ub (pSer65) G. Howeve15N-labeled Ub as an internal standard.Next we addressed the consequences of Ub phosphorylation for DUB activity and specificity. We purified dimers of Ub, dimers of Ub (pSer20), dimers of Ub (pSer57), and dimers of Ub (pSer65) from each linkage assembly reaction , yieldinWe tested the cleavage of each of the 27 purified Ub dimers with 31 DUBs in 837 Ub dimer/DUB combinations  and S4. DUBs did not cleave the unphosphorylated Ub dimer for 88 of the Ub dimer linkage/DUB combinations tested. Phosphorylation of Ub at Ser20, Ser57, or Ser65 did not activate DUB-mediated cleavage of the Ub dimers that were not cleaved when Ub was unmodified . For theOur data define the role of Ub phosphorylation at Ser20, Ser57, and Ser65 in regulating the DUB-mediated cleavage of each Ub linkage  and 6. CUSP8 cleaved K6-linked dimers of Ub (pSer20) and Ub (pSer57) more efficiently than K6-linked dimers of Ub, and phosphorylation of Ub at residue 65 substantially reduced K6 dimer cleavage by USP8 B and 6D.The cleavage of K11-linked Ub dimers by OTUD3 was activated by phosphorylation of Ser20 in Ub, but it was inhibited by phosphorylation at Ser57 or Ser65 C. USP4 aThe cleavage of K29-linked dimers by USP2 was activated by phosphorylation at Ser57, but the cleavage of K29-linked dimers of Ub by USP2 was deactivated by Ser65 phosphorylation D and 6E.The cleavage of K48-linked dimers of Ub by OTU family DUBs (OTUB1 and OTUB2) and several USP family DUBs  was activated by phosphorylation of Ser65, but it was deactivated by phosphorylation at Ser20 or Ser57 F and 6G.Our data also define how each Ub phosphorylation alters the specificity with which DUBs cleave different Ub linkages  and 6. SSer57 phosphorylation increased USP2-mediated cleavage of K29-linked Ub, but it decreased USP2-mediated cleavage of K6 and K48 linkages K. Ser57 Ser65 phosphorylation increased OTUB2-mediated cleavage of K48-linked Ub, but it decreased OTUB2-mediated cleavage of K63 linkages N. Ser65 We report the synthesis of multi-milligram quantities of Ub phosphorylated at position 20, 57, and 65 and the structure of Ub phosphorylated at Ser20. We report the assembly of K6-, K11-, K33-, K48-, and K63-linked Ub bearing phosphorylation at Ser20, Ser57, or Ser65 and the assembly of K29-linked Ub bearing Ser57 or Ser65 phosphorylation. While we have only sampled a small subset of possible E3 ligase activities on phosphoubiquitin species, our data demonstrate that the site-specific phosphorylation of Ub can control the extent and isomeric composition of Ub chains synthesized and that phosphorylation at distinct sites on Ub have distinct effects on Ub polymer synthesis. The most striking observation from the chain assembly experiments is the change in linkage specificity of UBE3C upon phosphorylation at Ser20 of Ub; this enzyme assembles K29 and K48 linkages with comparable efficiency when using Ub as a substrate, but it primarily assembles K48 chains with a Ub (pSer20) substrate B.UBE3C associates with the proteasome . In respOur data provide a wealth of new information on the consequences of Ub phosphorylation on DUB activity. Previous work investigated the effects of Ser65 phosphorylation on a single preferred substrate of five DUBs , which dDUBs bind to their substrates through a distal Ub-binding site (which binds the Ub that yields a free carboxylate upon [iso]peptide cleavage) and a proximal binding site (which yields a free amine upon cleavage) . The disThere are structural data for four DUBs with Ub in both the proximal and the distal binding sites  I–6O, theWe demonstrate that the cleavage of a Ub linkage type may be regulated by phosphorylation of Ub and that phosphorylation at different sites in Ub can have distinct effects on the cleavage of a linkage. Since phosphorylation of serine residues in Ub commonly inhibits DUB activity but has less effect on the subset of Ub chain synthesis enzymes tested, we suggest that phosphorylation may regulate the levels of Ub chains, primarily through effects on DUB activity and specificity.15N-Ub, and Ub oligomers were by standard methods  media containing 50 μg/ml chloramphenicol and 25 μg/ml tetracycline. The culture was diluted 1:100 into fresh TB media containing 25 μg/ml chloramphenicol and 12.5 μg/ml tetracycline and incubated at 37°C; once the OD600 reached 0.5, dexfosfoserine (pSer) (Bachem) was added to a final concentration of 2 mM. After 30 min of further incubation, additional pSer was added to the culture to reach a final concentration of 4 mM, and the culture was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 6 hr.BL21 ΔserB(DE3) cells containing pKW2 EF-Sep and pNHD-Ub (described in g for 30 min and filtration through a 0.4 μm polyethersulfone (PES) membrane. Ub was purified using nickel affinity chromatography  with a linear gradient of imidazole (30–500 mM). The protein was dialysed overnight against 10 mM Tris-HCl at 4°C, the C-terminal His6-tag was removed by incubating the sample with 15 μg/ml UCHL3 at 37°C for 5 hr, and Ub was further purified by ion exchange chromatography , using an NaCl gradient (0–500 mM) in 50 mM ammonium acetate (pH 4.5). Pure fractions were confirmed by ESI-MS and pooled before overnight dialysis against 20 mM Tris-HCl (pH 7.4). The sample was then concentrated to ∼15 mg/ml using Amicon Ultra-15 (3 kDa molecular weight cut off [MWCO]) centrifugal filter device (Millipore). All purifications were performed at 4°C.Cells were harvested by centrifugation, resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 30 mM imidazole, 0.5 mg/ml lysozyme (Sigma), and 50 μg/ml DNase (Sigma), and lysed by sonication. The lysate was clarified by centrifugation at 39,000 × 1UBQ [Purified Ub (pSer20) in 20 mM Tris (pH 7.4) was crystallized at 10 mg/ml in a hanging-drop setup using the vapor diffusion method. Crystals grew in 45% (w/v) PEG 400 and 100 mM Tris-HCl (pH 7.0), and they were vitrified in the same mother liquor. Data were collected at the ID-29 beamline at the European Synchrotron Radiation Facility (ESRF). The structure was determined by molecular replacement in Phaser , using a1UBQ ) that la15N-labelled Ub (pSer65). Y.A.K. provided reagents and advice for Ub chain purification together with Y.K. D.T.R performed initial cloning and expression of Ub(TAG) constructs with N.H.-D. All authors analyzed the data. N.H.-D. and J.W.C. wrote the paper with input from all authors.J.W.C. defined the direction of research. N.H.-D. designed and performed all biochemical experiments and performed crystallographic analyses. J.P. performed MS with AQUA peptide standards experiments and V.D.C. performed DUB experiments under the supervision of M.T. A.K. provided reagents for biochemical experiments and purified


Phosphorylation is the most widespread and well studied reversible posttranslational modification. Discovering tissue-specific preferences of phosphorylation sites is important as phosphorylation plays a role in regulating almost every cellular activity and disease state. Here we present a comprehensive analysis of global and tissue-specific sequence and structure properties of phosphorylation sites utilizing recent proteomics data. We identified tissue-specific motifs in both sequence and spatial environments of phosphorylation sites. Target site preferences of kinases across tissues indicate that, while many kinases mediate phosphorylation in all tissues, there are also kinases that exhibit more tissue-specific preferences which, notably, are not caused by tissue-specific kinase expression. We also demonstrate that many metabolic pathways are differentially regulated by phosphorylation in different tissues. Protein phosphorylation is a reversible posttranslational modification (PTM) that represents the most common PTM type in eukaryotes and plays a crucial role in many essential cellular processes, including cellular signaling, metabolism, differentiation, regulation of protein activity and subcellular localization . ProteinQuantitative mass spectrometry-based phosphoproteomics has allowed for a comprehensive characterization of serine/threonine/tyrosine phosphorylation sites, enabling the analyses of molecular mechanisms behind phosphorylation. Multiple attempts have been made to derive consensus sequence motifs of phosphorylation sites , 3, analRecent research indicates that many PTMs, including phosphorylation sites, are differential across tissues –24. In oet al. [Here we present a comprehensive sequence- and structure-based analysis of tissue-specific phosphorylation sites as well as global phosphorylation sites, employing a recent experimental dataset made available by Lundby et al. . We examWe used the dataset of 31480 phosphorylation sites in 7280 proteins identified by high-resolution tandem mass spectrometry in 14 rat tissues: brain , heart, muscle, lung, kidney, liver, stomach, pancreas, spleen, thymus, intestine, testis, perirenal fat, and blood . The UniThe best-matching UniProt ID for each phosphorylated peptide was identified as described previously . Brieflyet al. and matching them to those tissues in which the protein containing the reference site had at least one experimentally observed PSS/PTS/PYS. Then, the 21-mer sequences (from -10 to +10) surrounding each site in both positive and negative datasets were extracted and homology reduction on these 21-mers was performed using CD-HIT [We also generated a negative  dataset by extracting all S/T/Y residues not annotated as phosphorylated by Lundby g CD-HIT  at the 7We used the Two Sample Logo method  to find We applied the same procedure as in our previous study  to colleOnce the structure-based positive and negative PSSs/PTSs/PYSs datasets were generated as described above for the sequence-based data, homology reduction was again performed at the 90% identity level, taking into account only solvent accessible phosphorylation sites. The resulting dataset, which we call PS3D-90, contains 609 phosphorylation sites  and 10756 non-phosphorylation sites  in 332 proteins with known structures . The numThe R environment  was usedBy calculating the occurrence of 20 different amino acid types within the radial distances of 2 to 12 Å from the phosphorylated S/T/Y residue, and excluding amino acids already present in the sequence vicinity of PSSs/PTSs/PYSs, 3D and pure 3D environments of phosphorylation sites in the PS3D-90 dataset were determined, respectively. Distances between amino acid residues were defined based on the minimal distance between any pair of atoms belonging to these residues. For both types of environment the Fisher exact test was performed to assess the significance of the differences between PSSs/PTSs/PYSs and non-PSS/non-PTSs/non-PYSs in each tissue, and we used our in-house software tool to visualize these differences efficiently. We applied the procedure described in our previous work  to find We extracted structural features of phosphorylation sites as described previously . Brieflyclass and protein domain levels of the SCOP hierarchy [structural class level, the significance threshold of 0.05 was used, whereas at the protein domain level false discovery rate control was performed for multiple hypothesis correction in each tissue, and the significance threshold of 0.05 was used after all p-values were adjusted. Note that the numbers of PTSs and PYSs associated with known SCOP folds were not sufficient for comparison; therefore, they were excluded from this analysis.Structural folds of phosphorylated proteins from the PS3D-90 dataset in each tissue were examined according to the ierarchy . At the Using the best-matching UniProt identifiers of each PSSs/PTSs/PYSs and non-PSSs/non-PTSs/non-PYSs in the PS1D-70 dataset, pathways obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG) database  were anaet al. [In order to analyze enriched kinases across tissues, we used the substrate-matched kinase data given by Lundby et al.  where phet al. . This toRelationships between kinases, tissues and motifs were visualized by means of a tripartite graph as implemented in Cytoscape . Only tht-Test was used. The expression value of a particular kinase in a particular tissue was compared to expression values of the same kinase in all tissues. All p-values were adjusted and the significance threshold of 0.01 after the correction was used.For each kinase we identified the corresponding rat UniProt ID from the PhosphoSitePlus database , which cWe first examined global phosphorylation trends at the sequence level based on the PS1D-70 dataset. In general our findings are in line with previous studies , 39, 40.Previous studies have shown that beyond the general trends, phosphorylation sites exhibit kinase-specific sequence and spatial motifs as well as compartment-specific sequence motifs , 7, 21. We utilized the Motif-X software to identify enriched sequence motifs that are exclusively tissue-specific and cannot be detected when analyzing global trends , we performed a separate analysis of phosphorylation sequence motifs in the subset of proteins possessing a known 3D structure (dataset PS3D-90). The results obtained are markedly different from the ones derived from the PS1D-70 dataset due to the fact that structurally characterized proteins are depleted in disordered regions, which leads to a different amino acid composition. Specifically, negatively charged residues, as well as disorder-related glycine and serine residues are less pronounced in this dataset. For instance, aspartic acid and glutamic acid are not observed at position +1 of the global and tissue-specific PSSs Figs . Lysine Previous studies found only minor differences between the 3D structural surroundings of phosphorylated and non-phosphorylated sites, whereas stronger trends were observed when taking into account kinase preferences . In thisSimilar to the trend in the sequence motifs discussed above cysteine (C) residues are strongly avoided in the spatial surroundings of PSSs in all tissues. Strong enrichment of proline and aspartic acid residues is observed in close proximity of global PSSs , 27.05% (p < 6.9 x 10−6) and 30.02% (p < 1.5 x 10−3) on average, respectively  and systematic lupus erythematosus  and lysine deacetylases (KDACs) , the finferences . For ins Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Supporting Figures(DOCX)Click here for additional data file.S1 Supporting Tables(DOCX)Click here for additional data file.


Climate change has been predicted to reduce Antarctic sea ice but, instead, sea ice surrounding Antarctica has expanded over the past 30 years, albeit with contrasted regional changes. Here we report a recent extreme event in sea ice conditions in East Antarctica and investigate its consequences on a seabird community. In early 2014, the Dumont d'Urville Sea experienced the highest magnitude sea ice cover (76.8%) event on record ). Catastrophic effects were detected in the breeding output of all sympatric seabird species, with a total failure for two species. These results provide a new view crucial to predictive models of species abundance and distribution as to how extreme sea ice events might impact an entire community of top predators in polar marine ecosystems in a context of expanding sea ice in eastern Antarctica. IncreGiven the utmost importance of sea ice in the functioning of the Antarctic Ocean ecosystems –10, the Here, using data derived from a 50 year long-term observational study of top predator responses to climate change in the Southern Ocean, we report an unprecedented high sea ice concentration event and investigate its ecological consequences on an entire seabird community.3.3.1.http://iridl.ldeo.columbia.edu/SOURCES/.NOAA/.NCEP/.EMC/.CMB/.GLOBAL/.Reyn_SmithOIv2/.monthly/.sea_ice [k=30) using the ‘segmented’ package in R [k evenly spaced potential break points [The monthly sea ice concentration data for the Dumont d'Urville Sea area  were obtained from a suite of satellite-based passive microwave sensors provided by the National Snow and Ice Data Center and available at the International Research Institute for Climate and Society. This area corresponds to the main foraging area of the seabird species breeding at the Pointe Géologie archipelago during summer . Sea ice.sea_ice  for Januage in R ,18. Thisk points .3.2.′ S, 140°01′ E) situated on the edge of the Antarctic continent and adjacent to the Dumont d'Urville Sea since the early 1950s. Breeding success was defined as the proportion of eggs laid producing a fledging for species that lay a single egg and as the number of fledged chicks per breeding pair for two egg laying species. For southern fulmars, snow petrels and Wilson storm petrels nest sites were monitored and the numbers of eggs laid, chicks hatched and chicks fledged were recorded , together with a significant but slight sea ice loss during winter since the year 1992 . Following this summer trend in sea ice increase, the Dumont d'Urville Sea experienced in January and February 2014 unprecedented sea ice concentrations 473% above normal (Time series of summer (December–March) sea ice concentration exhibited a significant change towards more sea ice since 1982, with more rapid sea ice increase around the year 2011 : 2010, 2012; Davies tests, e normal . For thePygoscelis adeliae and Wilson's storm petrel Oceanites oceanicus). This resulted by far in the lowest average breeding success for the community , for one species most of the failures occurred during the chick stage (emperor penguin Aptenodytes forsteri: 67.5%), whereas for two other species failures occurred during both the egg and the chick stage .The entire seabird community exhibited a common response to the 2014 extreme event . All theommunity . For twoz=1.88, p=0.06). For the period 1982–2014, thus including the extreme sea ice concentration year of 2014, breeding success was negatively related to sea ice concentration in summer for most summer breeding species, but not for the only species that breeds in winter, the emperor penguin (Penguins had a lower success (0.055±0.055) than flying seabirds  were particularly affected, and the least affected species was the pagophilic snow petrel. Finally, extreme sea ice concentration may have increased protection from predators for prey species using sea ice as a refuge and which are commonly consumed by seabirds , thereby enhancing difficulties for predators to access food resources.Several lines of evidence strongly suggest that the extremely low breeding success of the seabird community was driven by extreme high sea ice concentrations. First, there is an association between most species' breeding success and sea ice concentration in Terre Adélie and in other sites in eastern Antarctica . Second,Importantly, models fitted to breeding success and summer sea ice concentration data, excluding the last year of unprecedented sea ice concentration (2014), predicted completely different responses to the 2014 high sea ice concentration compared with the observed responses. For example, although the model for the Adélie penguin breeding success had a good fit for the period 1982–2013 (R adjusted=0.509, These results are of primary interest for future studies investigating the ecological impacts of climate change and extreme events. Antarctic sea ice projections from general circulation models suggest a sea ice loss by the end of the twenty-first century ,35. Rece


The aim of this study was to quantify the inhibitory potency and binding affinity of a wide range of dietary polyphenols and determine the structural requirements for VEGF inhibition.250) of VEGF‐dependent VEGFR‐2 activation in HUVECS was determined after pretreating VEGF with polyphenols at various concentations. Binding affinities and binding sites on VEGF were predicted using in‐silico modelling. Ellagic acid and 15 flavonoids had IC50 values ≤10 μM while 28 other polyhenols were weak/non‐inhibitors. Structural features associated with potent inhibition included 3‐galloylation, C‐ring C2=C3, total OH, B‐ring catechol, C‐ring 3‐OH of flavonoids. Potency was not associated with polyphenol hydrophobicity. There was a strong correlation between potency of inhibition and binding affinities, and all polyphenols were predicted to bind to a region on VEGF involved in VEGFR‐2 binding.The concentration of polyphenol required to cause 50% inhibition (IC3Specific polyphenols bind directly to a discrete region of VEGF and inhibit VEGF signalling, and this potentially explains the associations between consumption of these polyphenols and CVD risk. BCAbicinchoninic acidCGcatechin gallatedp 2apple procyanidin oligomer degree of polymerisation 2dp 3apple procyanidin oligomer degree of polymerisation 3dp 4apple procyanidin oligomer degree of polymerisation 4ECGepicatechin gallateEGCepigallocatechinEGCGepigallocatechin gallateEGM‐2endothelial cell growth medium‐2HUVECshuman umbilical vein endothelial cellsrhVEGFrecombinant human vascular endothelial growth factorRIPA bufferradio‐immunoprecipitation assay bufferVEGFvascular endothelial growth factorVEGFR‐2vascular endothelial growth factor receptor 21Plants synthesise a range of polyphenols via the phenylpropanoid biosynthetic pathway and a range of these are consumed as part of human diets in the form of foods and beverages. Flavonoids make up about one third and other polyphenols (mainly simple phenolics) about two thirds of the polyphenols consumed in human diets, with estimates of average daily intakes being around 1200 mg per day −/− mice To date, the most scientifically robust evidence of health benefits accruing from consumption of dietary polyphenols centres on cardiovascular diseases. The biggest cause of cardiovascular disease is atherosclerosis which is a long‐term process in which artery walls become thickened through the formation of numerous atherosclerotic plaques. This process is dominated by the recruitment of white blood cells to artery walls and the inflammatory responses they produce, and is driven by accumulation of low‐density lipoproteins (which are taken up by the WBCs), differentiation of the white cells into macrophages (pro‐inflammatory) and insufficient removal of fats and cholesterol by high‐density lipoproteins. The growth of atherosclerotic plaques causes thickening of the vascular intima and a crucial consequence of this is to increase the thickness above that capable of supporting diffusion of oxygen and nutrients from the blood. This triggers angiogenesis and the formation of new blood vessels to supply the deeper parts of the plaque with oxygen and nutrients. Angiogenesis, the process resulting in the formation of new blood vessels from pre‐existing ones, plays an important role in the development and destabilisation of atherosclerotic plaques Recently, we reported that two flavan‐3‐ols , but not others like epicatechin, potently inhibited VEGF‐induced VEGFR‐2 phosphorylation in human umbilical vein endothelial cells (HUVECs), and that potent inhibition only occurred when the VEGF was pre‐incubated with the polyphenols The overall aim of the research reported here was to assess the ability of a wide range of polyphenols to bind to VEGF and consequently inhibit VEGF‐dependent VEGFR‐2 signalling, and to develop a better understanding of the structural and physicochemical features of polyphenols that contribute to them being potent or impotent inhibitors. We also determined binding affinities for all the polyphenol‐VEGF interactions, and the location of the binding site(s) on the VEGF macromolecule.22.12. All experiments were performed with HUVECs between passages 4 and 5.Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza , and maintained in Endothelial Cell Growth Medium‐2 (EGM‐2) (Lonza). The cells were cultured at 37ºC in an atmosphere at 5 % CO2.2The following polyphenols were purchased from Sigma  , Extrasynthese  , or Apin‐Chemicals  .Isolated single‐mass procyanidin fractions with various degrees of polymerisation  were isolated from apples as previously described 2.3The specific assay used to assess the potency with which polyphenols interact directly with VEGF and inhibit VEGF‐induced VEGFR‐2 phosphorylation has been described elsewhere 165; R&D Systems, Abingdon, UK) in endothelial basal medium (EGM‐2 with no serum or growth factors) for 5 min . Towards the end of the 5 min incubation period, confluent HUVECs were washed two times with warm PBS in preparation for the addition of the mixture of VEGF and polyphenol. Control incubations included a vehicle control  and VEGF  alone. After 5 min exposure of the HUVECs to the pre‐incubated treatments, cells were washed two times with ice‐cold PBS and then lysed with RIPA buffer (radio‐immunoprecipitation assay buffer) containing protease and phosphatase inhibitors. Lysates were transferred to eppendorf tubes and kept on ice for 15 min with periodic vortexing, and subsequently the lysates were clarified by centrifugation at 13 000 g for 10 min at 4°C. Supernatants were stored at –80°C until analysis. The total protein content of lysates was determined using a commercially available BCA assay .For each concentration tested, the polyphenol (stocks dissolved in DMSO) was incubated with VEGF  to assess if they had any significant inhibitory activity. Those polyphenols that significantly decreased the phosphorylation of VEGFR‐2 in HUVECs at 100 μM were pre‐incubated with VEGF at a range of concentrations (0.025–200 μM). The specific concentrations selected for each polyphenol were initially estimated from the extent of inhibition at 100 μM. If the inhibition of VEGFR‐2 activation was always less than 50% or always more than 50% then additional assays were conducted such that final datasets for IC2.450) and their confidence intervals were determined by using the log (inhibitor) versus normalised response – variable slope analysis tool in the GraphPad Prism software.Quantification of phosphorylated VEGFR‐2 in lysates was determined using a PathScan Phospho‐VEGFR‐2(Tyr1175) sandwich ELISA kit , following the manufacturer's instructions. The half inhibitory concentrations ‐catechin were very weak inhibitors  and (−)‐epicatechin was not an inhibitor at even the highest concentration tested , suggesting an important role for the gallic acid ester group in monomeric catechins. It has been reported that gallic acid esters of flavanols such as epigallocatechin (giving rise to EGCG) are potentially very unstable and degrade rapidly in physiological buffers 3‐Galloyl esters of (+)‐catechin and (−)‐epicatechin were potent inhibitors of VEGF‐mediated VEGFR‐2 activation, whereas the corresponding non‐galloylated compounds were very weak inhibitors  the C2═C3 double bound , (2) the 4‐oxo group  and (3) the positive charge (present in cyanidin), and the B‐ring position in C‐ring. Considering that all these compounds were hydroxylated in the 5‐, 7‐, 3′‐ and 4′‐ positions, the range of inhibitory efficacy was remarkably large, with the most potent being quercetin with an IC50 value of 0.75 μM to taxifolin which only inhibited VEGF activity by 12.3% at 100 μM ‐catechin, whose structure does not contain either the C2═C3 double bound or the 4‐oxo group  permit more twisting of the B‐ring in relation to the C‐ring, whereas a C2═C3 double bond increases the p‐conjugation of the bond linking the B‐ and C‐rings, which favors near‐planarity of the two rings. Molecules with near‐planar structures have been shown to more easily enter hydrophobic pockets in proteins 50 value for cyanidin was 200‐fold lower than (+)‐catechin (Table Although neither cyanidin or (+)‐catechin possess either the C2═C3 double bound or the 4‐oxo group in the C‐ring, the IC50>300‐fold lower than EGC, with the main structural difference being the lack of the C2═C3 double bound in conjugation with the 4‐oxo group compared with myricetin  , and the IC50 for tri‐hydroxylated EGC  was 42.9 μM while di‐hydroxylated epicatechin  was ineffective at 200 μM. Further, the order of potency for the tri‐, di‐, mono‐ and non‐hydroxylated B‐ring series of flavonols were as follows: myricetin  > quercetin  > kaempferol  > galangin . Similarly, the flavone luteolin  had a reasonably low IC50 of 7.46 μM whereas chrysin  inhibited only 58.6 % of VEGF‐induced VEGFR‐2 activation at 100 μM, and the same effects were observed for isoflavones [compare orobol  with genistein ]. VEGF inhibitory activity was affected not just by the number of hydroxyl groups in the B‐ring but also by the position of hydroxylation. For example, although quercetin  and morin  differ only in the position of the two hydroxyl groups on the B‐ring, the IC50 of morin was about 2‐fold higher than that of quercetin  showed IC3.4.250 value decreased significantly (and potency increased) as the number of OH group substitutions on the A‐ring increased. For example, quercetagetin  was a sevenfold more potent VEGF inhibitor than quercetin . There was also a strong effect of position of A‐ring hydroxyl groups. For example, luteolin  with OH groups in a meta arrangement potently inhibited VEGF activity (IC50 = 7.46 μM) while 3′,4′,7,8‐tetrahydroxyflavone with OH groups in an ortho position only inhibited VEGF activity by 63.2 % at 100 μM  was 10 times lower than that for luteolin   between the total number of hydroxyl substitutions and the potency of a flavonoid in terms of inhibition of VEGF‐induced VEGFR‐2 activation within the non‐gallated monomeric flavonoids ‐catechin: 5 OH groups, IC50 = 214.7 μM; rhamnetin: 4 OH groups, IC50 = 0.547 μM). As discussed above, 3‐galloylation substantially increases the potency of inhibition of flavan‐3‐ols and this may be related to the additional three hydroxyl groups this adds to the structure. Increasing the degree of polymerisation of flavan‐3‐ols increases the total number of hydroxyl groups and also increases their inhibition potency  variations in the number of hydroxyl substitutions on monomeric flavonoids, (ii) 3‐galloyl esterification of flavan‐3‐ols, and (iii) oligomerisation of flavanols. There was a positive correlation R = 0.98733.5Polyphenols are often found with substitution of hydroxyl groups. For example, in plants flavonoids are almost always found O‐linked to sugars through one or more hydroxyl positions on the A, B or C‐rings. Further, during absorption in humans, flavonoid glycosides are deglycosylated by human β‐glucosidases in the gastrointestinal tract 50 value than quercetin, which has a hydroxyl group on that position , modified the inhibition of VEGF‐induced VEGFR‐2 activation. All of the glycosides tested here  were completely ineffective at 50 μM. In addition, phloridzin, the 2′‐glucoside form of phloretin was completely ineffective and piceid (resveratrol‐3‐glucoside) inhibited VEGF activity by only 28% at 50 μM  of quercetin, which was one of the most potent inhibitors  and the capacity to inhibit VEGF‐dependent VEGFR‐2 activation (IC50)  Fig. B  between the predicted binding affinities and the experimentally determined IC50 vales for the set of 15 monomeric polyphenols that exhibited significant inhibitory activity . Remarkably, there was a very strong correlation ; the total number of hydroxyl groups on the B‐ring; the presence of the catechol group on the B‐ring; hydroxylation of position 3 on C‐ring; lack of further substitution of hydroxyl groups on the B‐ring. Inhibitory activity was not associated with polyphenol hydrophobicity, and the polyphenols were predicted to bind to a similar region of VEGF which is involved in interactions with its receptor VEGFR‐2, with binding affinities strongly correlated with inhibitory potency. These data are consistent with the notion that potent VEGF inhibitory activity of specific polyphenols is a consequence of specific binding of polyphenols to distinct binding sites on VEGF dimers. It is noteworthy that the polyphenols identified here to have the highest affinities to and to show the strongest inhibition of VEGF activity are those that have most commonly been reported to exhibit anti‐angiogenic and anti‐atherosclerotic activities in a range of in vitro models Data presented here show that some specific polyphenols are potent inhibitors of VEGF‐dependent VEGFR‐2 activation, with 11 polyphenols possessing ICThe authors have declared no conflicts of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting TableClick here for additional data file.


Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation.A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients  and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons.We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons.The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may be regulated by lithium treatment in first-episode psychosis. Increasing evidence indicates that transcription factor specificity protein 4 (SP4) may play a role in major psychiatric disorders. SP4 is a zinc finger transcription factor that binds to G-C/T rich sequences, regulating the expression of a large number of genes implicated in diverse biological functions . SP4 is Reduced N-Methyl-D-Aspartate (NMDA) receptor function has been proposed as one of the main contributors to symptom emergence in schizophrenia –16. NMDASP4 levels are increased in cerebellar granule neurons upon treatment with the mood stabilizer drug lithium . As noteBased on these previous observations, we hypothesized that SP4 S770 phosphorylation may be altered in peripheral blood mononuclear cells in first-episode psychosis patients. The main aim of this study was to investigate whether phosphorylation at SP4 S770 was increased in peripheral blood mononuclear cells in first-episode psychosis, how it related to SP4 protein abundance, and if this phosphorylation could be modulated by treatment with lithium. To test this possibility, we included a subgroup analysis based on the presence of lithium treatment in our human sample. Moreover, to further explore the effect on SP4 S770 phosphorylation of lithium and other prescribed drugs found in the FEP cohort, we tested the effect of lithium, valproic acid, and olanzapine  in an in vitro cell model.Fourteen first-episode psychosis patients from the acute psychiatric ward of Parc Sanitari Sant Joan de Deu and fourteen similar age-sex-healthy controls were recruited and evaluated in a previously described study . InclusiPeripheral blood mononuclear cells (PBMC) were obtained as described . 30 ml bCerebellar granule neurons were obtained from P6 rats  and cultured in 25mM KCl as previously described ; SP4 immunoblot analysis: mean = 0.06; percentile 75th = 0.12; minimum and maximum ; between both analysis: mean = 0.06; Percentile 75th = 0.10; minimum and maximum ). For cerebellar granule neuron experiments, pSP4 and SP4 values from the same protein extracts were determined; SP4 values were normalized to GAPDH and phospho-SP4/total SP4 ratio was calculated and normalized to untreated, control, conditions.For human samples, four independent western blots were prepared with the same amounts of the same protein extracts for each human subject; immunostaining for pSP4 and total SP4 were detected in two blots each and normalized to β-actin values from the same membrane. We calculated pSP4/SP4 ratio by referring the mean values of pSP4 immunoreactivity from two independent immunoblot analyses to the mean values of total SP4 immunoreactivity from other two independent immunoblot analyses. Both pSP4 and total SP4, normalized to β-actin, were referred to a standard sample to allow comparison of relative levels between different sets of samples and between repetitions. Total levels of SP4 were reported in . The coe2-test) for categorical variables were used to compare FEP and control groups, and FEP treatment subgroups. Bivariate analyses were carried out to detect association of our variables with potential confounding factors , using Mann–Whitney test for two groups, and Spearman or Pearson correlation for non parametric and parametric quantitative variables. For studies of treatment effect on pSP4/SP4 in cerebellar granule neuron cultures, all results represent the mean ± SEM from at least 3 independent experiments. ANOVA followed by Bonferroni’s post hoc test for multiple comparisons were performed where indicated. Statistical analysis was performed with GraphPad Prism version 5.00 and SPSS 17. All statistical tests were two-tailed except where otherwise indicated and significance level was set to 0.05.For the analysis of PBMC samples from first-episode psychosis and control subjects D’Agostino & Pearson omnibus normality test was carried out to test whether the variables followed a normal distribution. Outliers were detected where indicated using Pierce’s criterion (as simplified by Gould) . Non parThe demographic and clinical characteristics for healthy subjects and patients with first-episode psychosis were previously reported  and are We determined SP4 S770 phosphorylation levels in protein lysates from PBMC of 14 FEP subjects and 14 matched healthy controls . We calcWe further searched for an effect of lithium treatment on SP4 phosphorylation in these patients. We divided the FEP group into two treatment subgroups according to the presence or absence of lithium treatment. We found significantly reduced levels of pSP4/SP4 ratio in the lithium treated FEP group compared to the lithium untreated FEP group , . In contWe also investigated whether pSP4/SP4 ratio associated with the degree of severity measured by PANSS, CDS and YMRS scales in FEP patients. We found that pSP4/SP4 ratio did not correlate with the scores of any scale , suggesting that the observed increase in pSP4/SP4 is not influenced by symptom severity.We analyzed the influence of other demographic variables such as age and gender on the ratio of pSP4/SP4 in the control-FEP comparison, and in an intragroup analysis of FEP subjects designed to control for possible effects on pSP4/SP4 ratio of duration of the illness and daily antipsychotic dose. This analysis showed that pSP4/SP4 ratio is not significantly associated with any of the potential confounding factors .To further explore the effect of lithium and other drugs prescribed in the FEP cohort on SP4 phosphorylation, we used rat primary cultured cerebellar granule neurons as an in vitro cell model. We tested if lithium chloride, valproic acid or olanzapine, the most frequently prescribed antipsychotic in our cohort, influenced the levels of SP4 S770 phosphorylation in cultured cerebellar granule neurons. We found that only treatment with lithium chloride significantly decreased pSP4/SP4 ratio  ; while pOur study shows that phosphorylation of transcription factor SP4 at S770 is increased in peripheral blood mononuclear cells of first-episode psychosis patients, that lithium-treated patients have reduced SP4 phosphorylation levels and that lithium suppresses SP4 phosphorylation in an in vitro cell model. Thus, our findings suggest that SP4 phosphorylation is a potential biomarker for first-episode psychosis which may be regulated by lithium treatment.Our data adds further evidence for a role for SP4 phosphorylation at S770 in the post-translational regulation of SP4 in the context of psychiatric disorders. An increasing body of evidence indicates that SP4 transcription factor has a role in complex psychotic disorders through its regulation of a large network of genes, including components of NMDAR signaling pathways , 27, 28.Lithium has been successfully used as a mood stabilizer for the treatment of bipolar disorder for decades, and, to date, remains the most efficient drug for long-term preventive treatment , 24. In In peripheral lymphocytes from patients, total SP4 immunoreactivity was not significantly affected by lithium treatment, suggesting that the pathways regulating SP4 phosphorylation and protein abundance are independent in human lymphocytes. In contrast, we previously reported that, in cerebellar granule neurons, lithium partially stabilized SP4 protein, leading to significantly increased protein levels in non-depolarizing conditions . Thus ouAnalysis of biological parameters in first-episode psychosis is helpful to identify altered players that could be involved in the etiopathogenesis of psychotic disorders in the early stages. We have recently described an increase in SP4 phosphorylation in the postmortem cerebellum of bipolar disorder and schizophrenia patients . The finA few limitations need to be acknowledged. The sample size analysed here is small and the findings require replication in a larger sample of healthy subjects and FEP patients treated and untreated with lithium. In addition, our first-episode psychosis cohort is not drug naïve since the patients had been receiving antipsychotic treatment. Nevertheless, treatment duration was shorter than 6 months in all cases, and our statistical analysis did not show a correlation of SP4 phosphorylation with the daily dose of antipsychotics suggesting that these treatments may not influence our results. Moreover, our study of treatment effect on SP4 phosphorylation in cultured rat cerebellar neurons showed that SP4 phosphorylation levels were not affected by in vitro treatment with the antipsychotic olanzapine, the most representative antipsychotic in our cohort (n = 8). Despite these limitations, findings from this study may contribute towards a better understanding of the molecular mechanisms that underlie psychosis. Peripheral blood screening of SP4 phosphorylation levels may be a useful peripheral biomarker of psychosis and potentially regulated by lithium treatment in the early stages of psychosis.S1 Fig(A), total SP4 (B) and β-actin (A and B) immunoreactivity. N/A, not applicable.Immunoreactivity of phospho-SP4 S770 (pSP4), SP4 and β-actin was determined in the same protein extracts from peripheral blood mononuclear cells (PBMC) of control , and first-episode psychosis  individuals as previously reported . Images (TIF)Click here for additional data file.S2 Fig(A), valproic acid (VPA) (B) or olanzapine (OLZ) (C). Images show full size immunoblots from phospho-SP4 S770, total SP4, phospho-GSK3β S9 (a positive control for lithium treatment), phospho-Tau S396 (a positive control for VPA) (B), p42/44 MAPK (a positive control for OLZ) (C) and GAPDH immunoreactivity.Cerebellar granule neurons were treated with serum-free media containing 25mM KCl for two hours in the presence of the indicated concentrations of lithium chloride (TIF)Click here for additional data file.S1 TableFEP, first-episode psychosis; APd, antipsychotic dose; PANSS, Positive and Negative Syndrome Scale; CDS, Calgary Depression Scale; YMRS, Young Mania Rating Scale; ARP, aripripazole; QTP, quetiapine; Li, lithium; OLZ, olanzapine; RS, risperidone; VA, valproic acid; N/A, not applicable.(XLSX)Click here for additional data file.


Protein phosphorylation is a vital mechanism in the regulation of all processes in eukaryotic and prokaryotic cells. It is one of the most important of those post-translational modifications which allow proteins to reversibly change their enzymatic activity, cellular localization, oligomeric state, half-life and interaction partners.Protein phosphorylation is mainly used by bacteria to adapt to changes in their environment , but it is also used for intercellular communication  are normally used for tyrosine phosphorylation  their results clearly show that cross-talk does take place between the BY-kinase and the Hanks type Ser/Thr kinase interaction networks; (ii) new kinase substrates were found, including those involved in DNA replication, transcription regulation and cell division; and (iii) tyrosine kinases can be bound by several cytosolic or transmembrane modulators. One of the most interesting of these is MinD, a binding partner of kinase PtkA. They found that MinD modulates the kinase activity of PtkA in vitro and that MinD attracts PtkA to the cell poles. MinD is an ATPase which binds to the membrane as a dimer in its ATP-bound state through an amphiphatic helix. It attracts the cell division inhibitor MinC to the membrane and it also interacts with MinJ, which, in turn, interacts with DivIVA. DivIVA localizes to the negative curvature of the forming septum and persists at the cell poles together with the MinCDJ complex during vegetative growth. This mechanism appears to block asymmetric septation during vegetative growth. This blocking mechanism must be overridden before the first morphologically distinct stage of sporulation, the formation of a thin asymmetric septum, can occur.In a recent paper in i et al.  describeminD mutant cells, to be misplaced from its normal polar site  after asymmetric cell division (Ben Yehuda et al., The study by Shi et al.  providesThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AMPK complexes containing γ1, γ2 or γ3 subunit isoforms were generated by expression in human cells. They displayed differences in all three effects by which adenine nucleotides cause regulation, i.e. in allosteric activation, promotion of phosphorylation and inhibition of dephosphorylation 50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1  in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC PRKAA1 and PRKAA2, encoding α1 and α2; PRKAB1 and PRKAB2, encoding β1 and β2; and PRKAG1–PRKAG3, encoding γ1, γ2 and γ3 , but we were unable to discern any effect of ADP on Thr172 phosphorylation [effect (i)]. We also found that AMP was almost 10-fold more potent than ADP in inhibiting Thr172 dephosphorylation, suggesting that AMP may remain as the crucial signal 172, -β1,-α1, -α2  antibodi−1) to the medium for 48 h [HEK-293 cells expressing tetracycline-inducible human γ1, γ2 and γ3 with N-terminal FLAG tags were obtained by inserting the appropriate DNA into cells carrying a Flp recombinase target site with co-transfection of DNA encoding Flp recombinase; expression of the γ subunits was induced by adding tetracycline  containing 4.5 g/l glucose, 10% (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 200 μg/ml hygromycin and 15 μg/ml blasticidin. The medium was replaced with medium containing 1 g/l glucose, 16 h before the treatments described in the text.AMARA peptide substrate [Cell lysates were made using the rapid lysis procedure . Lysatesubstrate  was used2 was kept at a constant 4.8 mM excess over the concentration of ATP.AMPK was activated by incubating the cells in phenformin (10 mM) for 60 min prior to cell lysis. Cell lysates  were incubated with 50 μl of EZview Red anti-FLAG M2 affinity gel and immunoprecipitated and washed as described previously . The fin2  and ATP . Incubation was for 10 min at 30°C, after which the reactions were stopped by washing the IPs three times in Hepes assay buffer. They were resuspended in 400 μl of assay buffer and split for assays and analysis by Western blotting.Cell lysates  were incubated with 100 μl of EZview Red anti-FLAG M2 affinity gel, immunoprecipitated and washed as described previously . The IPs2 (9.8 mM) to activate PP2Cα; the reactions were then conducted in a shaking incubator at 30°C for 10 min. They were stopped by washing the beads three times in assay buffer, resuspending them in 400 μl of assay buffer and dividing them into aliquots for kinase assays and analysis by Western blotting.AMPK was activated by incubating the cells in phenformin (10 mM) for 60 min prior to cell lysis. Cell lysates  were incubated with 100 μl of EZview Red anti-FLAG M2 affinity gel beads at 4°C for 2 h on a roller mixer. The beads were washed twice with 1 ml of immunoprecipitation buffer and twice with 1 ml of Hepes assay buffer , resuspeFor analysis of proteins, SDS/PAGE was performed using precast Novex NuPAGE Bis-Tris 4–12% gradient polyacrylamide gels in the MOPS buffer system (Invitrogen). Proteins were transferred on to nitrocellulose membranes using the IBlot system (Life Technologies) and blocked in Li-Cor Odyssey blocking buffer for 1 h, and detection was performed using the appropriate secondary antibody coupled to IR680 or IR800 dye. Membranes were scanned using the Li-Cor Odyssey IR imager. For analysis of immunoprecipitations, membranes were sensitized using SuperSignal Western Blot Enhancer (Thermo Scientific) before blocking with Li-Cor Odyssey blocking buffer.Statistical significance was tested using GraphPad Prism 6, using tests for significance as specified in figure legends.We first characterized the HEK-293 cells stably expressing FLAG-tagged human γ1, γ2 or γ3 by Western blotting. n=4). Thus, HEK-293 cells primarily express α1, whereas β1 contributes approximately one-third and β2 approximately two-thirds of total AMPK activity in these cells.Coomassie Blue staining was not sufficiently sensitive to assess the composition of the γ1, γ2 or γ3 complexes that we immunoprecipitated using anti-FLAG antibodies, but we analysed them using Western blotting B. They e172, with the largest effects being on the γ2 complex. The Ca2+ ionophore A23187, which activates the alternative upstream kinase CaMKKβ, also activated and caused Thr172 phosphorylation with all three complexes, although in this case the largest effects were on the γ3 complex , where there was a marked phosphorylation of the downstream target acetyl-CoA carboxylase (ACC) despite the fact that any increases in Thr172 phosphorylation were negligible [172 phosphorylation in response to A-769662, which binds at a different site from AMP [172 dephosphorylation. STO-609 ; resultsgligible . The lacfrom AMP , suggest50) 15±5 μM; concentration of AMP giving half-maximal inhibition (IC50) 0.66±0.28 mM; γ2 complex, activation, 7.8±1.0-fold; EC50, 16±4 μM; IC50, 0.34±0.09 mM; γ3 complex, activation, 1.9±0.1-fold; EC50, 5.5±2.1 μM; IC50, 0.73±0.22 mM. Thus, at 200 μM ATP, activation was greatest with the γ2 complex (8-fold), was less with the γ1 complex (4-fold) and was smallest (1.9-fold) with the γ3 complex. When the assays were conducted with 5 mM ATP the following parameters were obtained: γ1 complex, activation, 10±2-fold; EC50, 120±20 μM; IC50, 22±5 mM; γ2 complex, activation, 9±1-fold; EC50, 160±30 μM; IC50, 29±6 mM; γ3 complex, activation, 1.4±0.03-fold; EC50, 12±5 μM; IC50, 29±6 mM. Thus, when measured at this more physiological concentration of ATP, allosteric activation by AMP of both the γ1 and the γ2 complexes increased to ∼10-fold, whereas activation of the γ3 complex became almost negligible (1.4-fold). In addition, the EC50 values for allosteric activation by AMP of the γ1 and γ2 complexes were ∼10-fold higher at the higher ATP concentration , whereas the IC50 values for inhibition were 30–100-fold higher . This reflects the fact, as shown previously with rat liver AMPK [We next addressed the allosteric activation of complexes containing the different γ isoforms, using AMPK immunoprecipitated from HEK-293 cell stably expressing FLAG-tagged γ1, γ2 or γ3. These assays were initially performed both at the standard  ATP concentration of 200 μM A and at 50, 7.0±1.3 μM; IC50, 0.58±0.09 mM; γ2, activation, 3.5±0.2-fold; EC50, 4.2±1.2 μM; IC50, 0.65±0.15 mM; γ3, activation, 2.0±0.1-fold; EC50, 1.4±0.5 μM; IC50, 1.2±0.3 mM.Since there was more activation of the γ3 complex at the lower ATP concentration (200 μM), we repeated the assays in the presence of ATP concentrations 10-fold lower again (20 μM). Under these conditions C, maxima2+–Sepharose and phosphorylated Thr172 using CaMKKβ. 50, 16±1 μM; α1β1γ1: activation, 3.2±0.1-fold; EC50, 83±7 μM). However, the α2β2γ3 complex was barely activated at all (<15%) by AMP; this is similar to the results we obtained with γ1 and γ3 complexes expressed in HEK-293 cells  to remove phosphate from Thr172, were washed to remove PP1γ, and okadaic acid was added to block the activity of any residual phosphatase. They were then treated with limiting quantities of LKB1 in the presence of 5 mM ATP , with or without 1 mM AMP. This revealed .We next tested whether AMP promoted Thrrevealed A that AMrevealed B shows t172 phosphorylation by CaMMKβ as well as LKB1, as proposed previously [172 phosphorylation and activation to that of LKB1. 172 phosphorylation, with smaller effects on the γ2 and γ3 complexes, as in 172 phosphorylation by CaMKKβ using any of the γ subunit isoforms.We also tested whether AMP promoted AMPK activation and Threviously . The exp172 phosphorylation by LKB1 [172 phosphorylation with the γ1 complex and smaller effects with the γ2 and γ3 complex and all of these effects were abolished by the inclusion of CD73 in the assays as expected. ADP also appeared to cause a small stimulatory effect with the γ1 complex, although any effects on the γ2 and γ3 complexes were not significant. Unexpectedly, however, CD73 did not completely abolish the small stimulatory effect of ADP on the γ1 complex, suggesting that the effect was at least partly caused by binding of ADP, rather than by breakdown of ADP to AMP during the assay. The Western blots in 172 phosphorylation induced by LKB1 was completely abolished by addition of CD73, whereas the stimulatory effect of ADP was only partly abolished. Any effects of ADP on Thr172 phosphorylation by LKB1 of the γ2 and γ3 complexes were not detectable, although there were small effects of AMP that were abolished by CD73.Using native AMPK purified from rat liver (a mixture of the α1β1γ1 and α2β1γ1 complexes), we previously reported small effects of ADP on activation and Thr by LKB1 . However172 phosphorylation assessed by Western blotting in the same experiment. Concentrations of ADP from 0.1 to 1 mM caused a small stimulatory effect, but above 1 mM ADP caused a marked inhibition. It was not possible to obtain precise estimates of the EC50 for the activating effect or the IC50 for the inhibitory effect, because the ranges of concentrations over which they occurred were so similar. However, the EC50 for the activating effect of ADP appeared to be at least 1 mM, which is an order of magnitude higher than the EC50 for the effect of AMP (130 μM).172 phosphorylation, the complexes were then isolated by immunoprecipitation and equal amounts (based on anti-FLAG blots) were treated with a limiting quantity of PP2Cα in the presence of increasing concentrations of AMP or ADP. As before, in order to simulate physiological conditions, these assays were performed in the presence of 5 mM ATP. The upper panel of 172 antibodies (lower panel). The effects of ADP are shown in 50 values: 170±30 μM (AMP) and 910±70 (ADP) for γ1 and 120±30 μM (AMP) and 980±150 (ADP) for γ3. However, a striking difference with the γ2 complex was that ADP was almost equally potent in protecting against inactivation and Thr172 dephosphorylation as AMP. In this case, the IC50 values were 130±20 μM (AMP) and 180±20 (ADP). The upper panel of 172 blots (lower panel).Finally, we addressed the effects of AMP and ADP on the dephosphorylation of γ1, γ2 and γ3 complexes by the protein phosphatase PP2Cα. Cells expressing γ1, γ2 or γ3 complexes were treated with phenformin to promote Thr(i) Complexes containing γ1 and γ2 were allosterically activated by AMP up to 10-fold, with half-maximal effects (measured in the presence of 5 mM ATP) at 120 (γ1) and 160 μM (γ2), which are 30–40-fold lower than the concentration of ATP utilized in the assay (5 mM). By contrast, γ3 complexes were only significantly allosterically activated by AMP (2-fold) at very low ATP concentrations (20 μM) that are unlikely to be physiologically relevant.172 phosphorylation was stimulated >4-fold by AMP when LKB1, but not CaMKKβ, was the upstream kinase. Much smaller (<2-fold), although still significant, effects of AMP were observed using γ2 and γ3 complexes. The half-maximal effect of AMP on activation of γ1 complexes by LKB1, measured at 5 mM ATP (130 μM), was very similar to the half-maximal effect for allosteric activation (120 μM).(ii) Activation of γ1 complexes caused by Thr172 phosphorylation by LKB1 was also stimulated ∼2-fold by ADP, although we did not observe this effect with γ2 or γ3 complexes. Despite our earlier findings with native AMPK from rat liver [(iii) Activation of γ1 complexes caused by Thrat liver , this ef172 dephosphorylation, catalysed by PP2Cα, was reduced by AMP binding. The concentrations of AMP causing half-maximal effects, measured at 5 mM ATP, were similar for all three complexes (120–170 μM) and were similar to the concentrations causing half-maximal allosteric activation with γ1 and γ2 complexes and half-maximal activation of phosphorylation/activation by LKB1 using γ1 complexes. All of these effects were measured in the presence of a physiologically relevant concentration of ATP (5 mM).(iv) Inactivation of all three γ complexes by Thr172 dephosphorylation catalysed by PP2Cα was also protected by binding of ADP. Interestingly, however, the concentrations causing half-maximal effects, measured at 5 mM ATP, were similar for AMP and ADP using γ2 complexes (130 and 180 μM), but those for ADP were almost an order of magnitude higher than those for AMP using γ1 and γ3 complexes. Thus AMP is in general a more potent activator of AMPK than ADP, with the interesting exception of the γ2 complex.(v) Inactivation of all three γ complexes by ThrOur results clearly demonstrate that human AMPK complexes containing the γ1, γ2 or γ3 isoforms, when expressed in human cells, differ in several respects in their responses to adenine nucleotide. These differences can be summarized as follows:172 phosphorylation rather than any allosteric effect. It is indeed notable that the complex that is activated by muscle contraction is not subject to allosteric activation.The AMPK activity in HEK-293 cells is largely accounted for by the α1, β1, β2 and γ1 isoforms , althoug172 phosphorylation) that occurs in mammalian cells but not in bacteria. Since the regulation of the bacterially expressed complexes is in that sense abnormal, we chose to study the other regulatory mechanisms only with the complexes expressed in human cells.It remains unclear as to why the allosteric activation by AMP of γ1 and γ2 complexes is much greater for those expressed in mammalian cells rather than in bacteria, although we suspect that it may be due to some covalent modification  by CaMKKβ [172 phosphorylation might then have been caused by the ability of AMP to inhibit dephosphorylation. We avoided this potential problem by dephosphorylating AMPK in the IP using PP1γ rather than PP2Cα, by washing the precipitates extensively and by adding the PPP family phosphatase inhibitor, okadaic acid [What is the reason for the discrepancy between our results, in which AMP binding did not promote activation and Thry CaMKKβ ? One posy CaMKKβ . Althougaic acid , to the The molecular mechanisms for the differential effects of AMP, ADP and ATP on AMPK complexes containing the γ1, γ2 and γ3 isoforms remain unclear at present. All existing crystal structures for core ,26 or fu