Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered “Unique” domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes. The Src kinase family is composed of 10 proteins: Src, Frk, Lck, Lyn, Blk, Hck, Fyn, Yrk, Fgr, and Yes. Src family kinases (SFKs) are membrane-associated, non-receptor tyrosine kinases that act as important signaling intermediaries regulating a variety of outputs, such as cell proliferation, differentiation, apoptosis, migration, and metabolism. All SFKs share the same domain arrangement: a large catalytic C-terminal domain is preceded by regulatory Src 2 and 3 homology domains  and the membrane-anchoring SH4 N-terminal region, which contain myristoylation and palmitoylation sites as well as positively charged residues. The SH3 and SH4 domains are linked by an intrinsically disordered segment of 50–90 residues, called the Unique domain (UD) because of the lack of sequence similarity among the different SFKs. However, the UD of each individual SFK member is well conserved between different organisms suggesting a more specific role than that of a simple spacer. Figure Efforts to find specific UD functions during the last two decades have confirmed an active role of the UD in the regulation of SFK members. In the case of Lck, the UD mediates association with the cytoplasmic tail of CD4 and CD8α through a zinc clasp structure . For mosThe involvement of SFK signaling in growth, motility and cell survival makes them important oncology targets. Activation of SFKs is tightly regulated in healthy cells , while tThe UD is an intrinsically disordered region (IDR). Intrinsically disordered proteins (IDPs) are relatively more prevalent among signaling and cancer-related proteins . The conWhile the catalytic and Src homology domains of SFKs have long been subjects of investigation, the study of the regulatory mechanisms involving the intrinsically disordered UD has only recently started gaining momentum. Phosphorylation events are often associated with the regulation of important functional regions. In this mini-review we discuss the current knowledge about the phosphorylation events taking place in the Unique domains of SFKs.56PPASP60). in vivo mitotic phosphorylation in Lck  is a potential drug target for cancer and HIV infections. High levels of Hck are associated with drug resistance in chronic myeloid leukemia and Hck activity has been connected with HIV-1 . An impoLyn, at Tyr32, which then phosphorylates MCM7, a licensing factor critical for DNA replication. Phosphorylation at Tyr600 of MCM7 increases its association with other minichromosome maintenance complex proteins, thereby promoting DNA synthesis complex assembly and cell proliferation. Both p56Lyn Tyr32 and MCM7 Tyr600 phosphorylation are enhanced in proliferating cells and correlated with poor survival of breast cancer patients  β-receptor by binding to PDGF leads to activation of a member of the SFK . The UniSerine 21 within the UD of Fyn is part of a RxxS motif targeted by protein kinase A (PKA). Mutation of Ser21 to Alanine (Ser21Ala-Fyn) blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity . In the Protein kinase A is a crucial component of integrin-mediated signaling pathways. Interaction of cells with their substrate or adhesion to other cells leads to activation of PKA , and indSrc is a non-receptor protein tyrosine kinase with a key role in regulating cell-to-matrix adhesion, migration, and junctional stability . Thus, pAlthough the phosphorylation of Src at Ser17 by PKA (cAMP-dependent protein kinase) is a well-characterized process, its biological significance remains unclear . PotentiInteraction of Src with lipids is not restricted to the SH4 domain Figure . The uniXenopus laevis oocytes. Mutations abolishing lipid binding by the ULBR but not affecting the SH4 domain resulted in a conditional lethal phenotype after progesterone induced maturation  as well as Thr46 in chicken Src (with no correspondence in humans). These residues are phosphorylated by cyclin-dependent kinase 1 (Cdk1/Cdc2) during mitosis. These phosphorylations were found to activate Src by disrupting the interaction between the SH2 domain and Tyr527/Tyr530 (chicken/human) and facilitating the dephosphorylation by protein tyrosine phosphatases .In addition (or possibly related to) its effect on lipid binding, phosphorylation of Ser75 is important in other aspects of Src regulation. Mitosis-independent phosphorylation of this site was observed in neurons and in certain cancer cell lines; this phosphorylation was shown to be due to Cdk5, a widely distributed proline-directed kinase with a substrate specificity similar to Cdk1 . Active X. laevis egg extracts showed the phosphorylation of Ser75 and Ser69, as well as Ser 17  in disordered domains anchored to membranes by a second, more stable, binding site close to the protein termini (the SH4 domain in the case of SFKs) can modulate the position of the active domains (the kinase or regulatory domains in the case of SFKs) with respect to the membrane surface. Lipid binding by the internal site can switch off the access of the active domains to target sites of membrane-anchored substrates/partners located beyond a particular distance from the membrane surface or, conversely, facilitate the interaction with sites near the membrane surface. Phosphorylation or other modifications preventing the interaction of the internal site would result in a release of the active domains, which can then reach sites located further apart from the membrane surface or disfavor the interaction with membrane-proximal sites. This represents a new compartmentalization mechanism based on the relative position of interacting sites with respect to the membrane surface. This mechanism enables modulating the functional interactions between two proteins anchored next to each other in the membrane.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using  The discovery that constitutive ABL kinase activity is sufficient and necessary to cause CML laid the foundation for development of imatinib as a target-directed therapy123567Myeloproliferative neoplasms (MPNs) are a group of hematologic malignancies that include PhJAK2 is a cytosolic tyrosine kinase activated by cytokine-mediated receptor dimerization, resulting in phosphorylation of STATs required for cell proliferation, survival and myeloid development, as well as for the initial stages of the immune response1213131516The JAK2 inhibitor ruxolitinib was recently approved for the treatment of MF and PV, and numerous other inhibitors are in phase-II/-III clinical trials3192022We performed an unbiased chemical-genetic screen using two different JAK2 inhibitors, ruxolitinib and fedratinib, to identify a comprehensive set of drug-resistant variants, in order to glean regulatory mechanisms of resistance. Our screen identified 211 resistance mutations against ruxolitinib, but a complete lack of resistance against fedratinib. The resistance mutations conferred cross-resistance to other JAK2 inhibitors—AZD1480, CYT-387 and lestaurtinib, but failed to confer resistance against fedratinib. Biochemical characterization and structural modeling revealed that fedratinib simultaneously binds to both ATP-binding and peptide-substrate-binding sites, thereby preventing emergence of resistant clones.In vitro characterization of these clones showed both higher IC50 and increased resistance to ruxolitinib, thus suggesting that the BaF3-MPL cells expressing JAK2-V617F are not addicted to JAK2 because MPL overexpression seemingly bypasses the JAK2 dependent survival. Therefore, we performed screening using parental BaF3 cells transduced with randomly mutagenized JAK2-V617F and two clinically relevant JAK2 inhibitors: ruxolitinib and fedratinib. Ruxolitinib-resistant clones emerged at 1, 2 and 5 μM inhibitor—representing 10−, 25− and 50-fold increases in IC50 values for JAK2-V617F (~100 nM), respectively (We performed a ruxolitinib resistant screen using BaF3-MPL cells that showed emergence of resistant clones (data not shown). Although these clones conferred robust resistance to ruxolitinib, sequencing did not reveal mutations. ectively . In contectively .We next mapped resistant mutations onto crystal structures of JAK2 kinase domains28To verify the activity of individual mutations (since some resistant clones carried two or more mutations) and to elucidate the mechanisms of resistance, we generated 40 different variants in the FERM, SH2, JH2, and kinase domains of JAK2-V617F, using site-directed mutagenesis. Dose-response analysis revealed that all of these mutants are resistant to drug inhibition, except variant D976N . Three r30In contrast, variants M181R, R971G, L983F, E1006K, E1046K, and R1063P showed higher resistance when the receptors were activated by exogenous cytokines ; this su2826(i), loss of hydrogen-bond for anchoring, by substituting threonine for Asn 981, while variants E930G and L932F may disrupt hydrogen bond interactions perhaps by altering the dynamics of hinge region ((ii) steric hindrance  ((iii) active-site destabilization, by mutations in the kinase hinge , the catalytic loop (N981I/T) and the DFG motif (D994N) . Accordingly, many mutations clustered in the active site  were resistant, but did not exhibit any change in IC50 values when assayed in the presence or absence of IL-3 , or in Je region ; (ii) std L983F) ; and (ii (D994N) –7. Among (D994N) . In cell (D994N) . Structu (D994N) –C, sugge (D994N) , also su (D994N) . Likewis (D994N) , suggestBecause each inhibitor binds a unique conformational state, engaging different sets of amino acid residuesAll ruxolitinib-resistant JAK2 variants showed a complete lack of resistance to fedratinib, particularly when receptor was activated by IL-3 . This suTo better understand the molecular mechanism of fedratinib inhibition, we performed fedratinib molecular-docking experiments, by targeted docking to the ATP-site, substrate site and blind docking to the whole kinase domain (PDB:2B7A) by SWissDockin silico docking observation. These experiments demonstrated that ruxolitinib binds only the ATP site, while fedratinib binds both ATP and substrate-binding sites.To determine whether fedratinib inhibits by binding the ATP site or the peptide substrate-site, we performed structural modeling of two resistant ATP-site variants, Y931C and L983F, . These vmax. One principal mechanism for developing resistance against ATP-competitive inhibitors is to select for kinase activating mutations. In some instances these activating mutations may change the Km for ATP providing additional support to compete out inhibitor binding; a common mechanism exploited by gatekeeper mutations to confer resistance. To determine the kinetic parameters we expressed full length JAK2-V617F and seven ruxolitinib resistant variants in insect cells, proteins were purified using Strep-Tactin resins (m (ATP)app and Vmax , G417P (SH2 domain), D544H (SH2-pseudokinase linker) and Y931C (Kinase domain) displayed ~2 fold increase Vmax compared to JAK2-V617F. Interestingly variants L551P (pseudokinase domain) and Y931C (kinase domain) have reduced Km (ATP)app . However, resistant variant L983F did not show any significant change in Km (ATP)app and Vmax. Whereas, a kinetic analysis of purified kinase domain displayed ~20 fold (Y931C), 4 fold (E930G) and 2 fold (L983F) increase in Vmax with significant changes in Km (ATP)app by Y931C and L983F . Thermophoresis measures the motion of molecules in microscopic temperature gradients that allows measuring the affinity constants and binding stoichiometry of a wide variety of interactions in the binding equilibrium. We reasoned that if fedratinib binds to two different sites i. e. the ATP and the substrate site, the use of an ATP site mutant for binding analysis will show only one binding site. As predicted, fedratinib showed two binding sites in JAK2-WT with binding affinities having Kd values of 8307 nM (Site –I/ATP site) and 21 nM (Site-II/substrate site), To further probe the mechanism of fedratinib binding to the substrate-binding site, we created substrate-site mutations in the background of JAK2-V617F-Y931C by introducing bulkier, hydrophobic amino acids proximal to the inhibitor-binding site. We used the JAK2-V617F-Y931C, because blocking one binding site is probably insufficient to observe resistance conferred by a secondary site. As expected, mutations I1018F/W and L1026F reduced kinase activity, while F1019L, W1020C and Y1021H resulted in inactive kinase . I1018F/This study provides clear evidence for fedratinib binding to the substrate-binding pocket of JAK2 kinase. The data also suggest that dual binding of fedratinib—to both ATP and substrate-binding sites—prevents the emergence of resistant clones. These results help explain why fedratinib-mediated inhibition is greater when receptor is activated by cytokines—since growth factor-mediated receptor dimerization activates and stabilizes an active kinase conformation that allows easy access to the substrate-binding site. Altogether, these data suggest that mutations in the substrate-binding site would negatively affect the catalytic efficiency that will result in weak proliferation compared to normal cells. Thus these clones will not survive long, and, therefore, will be unable to confer resistance.in vitro screen  conferred high-grade resistance to all tested ATP-site inhibitors. To test whether they confer resistance in vivo, as a proof of concept, we transplanted Balb/c mice with 1 million BaF3 cells expressing luciferase and JAK2-V617F variants. As expected, twice daily administration of 100 mg/kg ruxolitinib eradicated the JAK2-V617F- and JAK2-V617F/Y931C-expressing cells  are susceptible to resistance. However, in contrast, fedratinib showed a complete lack of genetic resistance due to its unique binding to both ATP and substrate-binding sites. Enzyme kinetics, max (4.4 μM) for ruxolitinib at 100 mg/kgRecent clinical studies of JAK inhibitors suggest that these inhibitors are effective in relieving splenomegaly and constitutional symptoms of myelofibrosis, perhaps by modulating the inflammatory cytokines18Drug resistance to kinase inhibitors poses a great challenge for effective therapeutic response. One strategy to combat resistance is to design new ATP-site inhibitors that derive selectivity by alternative modes of binding, such as with BCR-ABL inhibitors dasatinib53In conclusion, we demonstrate that like other kinases JAK2 is prone to develop resistant mutations against ATP site inhibitors, but targeting the substrate-binding site prevents the selection of resistant mutations. This study provides a proof-of-concept for the therapeutic targeting of substrate-binding site of oncogenic kinases and establishes the rationale for clinical evaluation of this concept.The study involving mice conformed to the National Institutes of Health guide for the care and use of laboratory animals and was approved by the Cincinnati Children’s Hospital Institutional Animal Care and Use Committee (IACUC). The study involving human participants was approved by the institutional review board (IRB) of Cincinnati Children’s hospital. The methods were carried out in accordance with the approved guidelines. All clinical research was performed on the basis of the principles expressed in the declaration of Helsinki.Plasmid constructs pMSCV-JAK2V617F-puro and pMSCV-JAK2-puro were provided by Dr. Ross Levine, and pMSCV-TEL-JAK2-Ires-GFP, pMSCV-TEL-JAK1-Ires-GFP, pMSCV-TEL-JAK3-IresTo generate random mutation library of Jak2-V617F, E. coli XL1-red cells were transformed with one μg of JAK2-V617F plasmid DNA. More specifically, 50–100 ng of DNA (more than 100 ng of DNA decreases transformation efficiency) was mixed with 100 μl of competent cells in a pre-chilled polypropylene tube and incubated on ice for 30 min, gently swirling every 5 min. For a good library, four to six tubes of competent cells are used. Cells from each tube were plated on four 10 cm LB-agar plates containing 100-μg/ml ampicillin. Plates were incubated at 37 °C for 18–24 hours. When colonies became visible (18–24 hrs), cells were pooled from each plates and plasmid DNA was isolated using the Qiagen’s midiprep kit. At this stage, the heterogeneity of mutations in the library can be roughly assessed by restriction digestion with a frequent cutter such as Sau3A1 or Taq1 or sequencing.6. We did not used BaF3 cells expressing homodimeric receptor such as EPOR and Mpl, which has been used in other studies for screening. We observed that the overexpression of EPOR or MPL tend to loose dependence on Jak2 resulting to emergence of false-positives6 cells were mixed with 100 ml of virus supernatant and 100 ml of RPMI, containing 10% serum and 24 μg/ml of polyberene. These cell mixers centrifuged at 2500 rpm for 90 minutes at 37 °C. After centrifugation, the plates were transferred to incubator for 24 hour incubation at 37 °C. Next day, cells were pooled together and mixed with ruxolitinib and medium containing soft-agar plated in six well plates Plates were incubated at 37 °C for two weeks. Resistant colonies were picked and sub-cultured individually in 24 well plates for 3–4 days.To select resistant clones of JAK2-V617F, high titer retroviruses were produced as describeExpanded colonies were harvested 7 to 14 days after isolation from agar, whole genomic DNA extracted using the DNeasy genomic , and whole (3.5 kB) JAK2 was amplified by PCR using forward and reverse primers  and long template high-fidelity polymerase . Coding region was next sequenced by 8 different primers spanning the full coding region: P1, 5′-CGACGGCCAGTCTTAAGCTCG-3′; P2, 5′-GTAGTGGCAGCAGCAGAACC-3′; P3, 5′-GGAATTCAGTGGTCAAG-3′; P4, 5′-GAGAATACAACCTCAGCG-3′; P5, 5′-GATCACTGGATACATACC-3′; P6, 5′-GCATGGACTATGAGCCAG-3′; P7, 5′-CATATGGAAGTTTACGAG-3′; and P8, 5′-TGGAGTCTGGTCTTTCTCC-3′. Sequence alignment was performed using the Lasergene Genomics Suite (DNASTAR).Mutants isolated in the screen were engineered into pMSCV-JAK2-V617F-Puro, using QuikChange XL mutagenesis kit (Stratagene). Each point mutants were confirmed by sequence analysis.4 cell per well) in RPMI medium containing 10% FCS and lacking IL-3. Kinase inhibitors were included in the media at varying concentrations. After 60 hours, the number of viable cells was assessed using WST-1 reagent , according to the manufacturer’s specifications. Assays were performed in quadruplicate. Absorbance measurements at 450 nm were averaged and plotted against inhibitor concentration as a best-fit sigmoidal curve, using the Origin 7.0 non-linear curve-fitting algorithm ; the concentration resulting in 50% maximal inhibition was reported as the cellular IC50. BCR-ABL kinase assay and immunoblotting were performed as previously described28Ba/F3 cells expressing mutant BCR-ABL proteins were plated in 96-well plates  simulations of receptor-ligand complexes were performed using the AMBER package652 to constrain the motion of atoms of the protein-ligand complex but allowing to move water molecules freely. For the temperature equilibration, the temperature was raised gradually from 0 K to 300 K in three phases: (i) from 0 K to 50 K, (ii) from 50 K to 150 K and (iii) from 150 K to 300 K at constant volume. There were 200000 steps of a 200 ps MD simulation with time step of 0.001 ps. A force constant of the harmonic potential was 10 kcal/mol Å2 on solute atoms. For the equilibration of the solvent density at constant temperature 300 K and pressure of 1 atm, a 200 ps MD simulation with Langevin dynamics of collision frequency 1 ps−1 was run in 200000 steps. Finally, in the production runs 50 ns MD simulations at constant temperature of 300 K and a constant volume were performed for each of the two binding sites and the corresponding complex with the ligand either in ATP or substrate-binding pocket. For each of the sites, 50,000 snapshots of the complex were obtained and analyzed with AMBERTOOL14, xmgrace and awk. In the simulation, sander and PMEMD were used with MPI run and CUDA as needed in GPU environment.A minimization of 10,000 steps were done applying a harmonic potential of force constant 100 kcal/mol ÅSpodoptera frugiperda, Sf9 cells. Full-length Jak2-V617F and kinase domain having Strep-tags (WSHPQFEK) at c-terminus were cloned in pENTR222 (Invitrogen) using polymerase chain reaction (PCR). Point mutations in to the full-length Jak2-V617F  and kinase domains  were created by site directed mutagenesis using QuikChange XL mutagenesis kit. Each point mutants was confirmed by sequence analysis. These pENTR-JAK2-V61F and pENTR-JAK2-JH1 constructs were used to develop baculoviruses using Baculodirect kit (Invitrogen), according to manufacturer’s instructions. A high titer virus (109–1010 pfu/ml) was generated for each construct for protein expression. Three hundred mls of suspension culture of insect cells at a density of 2–3 × 106/ml were infected with viruses at 5-10 MOI (multiplicity of infection). Infection media was supplemented with 10% FBS as in the absence of serum expression is extremely low and often beyond detection level by western blotting. After 60 hrs of infection, cells were harvested for protein purification. All purification steps were carried out at 4 °C. The cells were lysed in lysis buffer . Cell lysates were incubated with Strep-Tactin resin (IBA) for two hrs at 4C, followed with batch purification. The JAK2 proteins were eluted in elution buffer . Elution buffer was exchanged with storage buffer  by Amicon ultrafilters.Proteins were expressed in Protein concentration and purity was determined by Bradford assay and SDS-PAGE analysis, respectively.Kinase assays were performed in triplicate, using the ADP-Glo kinase-assay kit , recombinant JAK2 purified from insect cells , and STAT5-derived peptide (AKAADGYVKPQIKQVV) as a substrate (synthesized by Genscript Inc). These assays were performed in triplicate in 96-well plates. For the ATP competitive assay, for each reaction, 50 ng of recombinant JAK2 was incubated with a graded concentration of inhibitor and 50 μM STAT5 peptide at different ATP concentrations . For the substrate competitive assay, 50 ng JAK2 was incubated with 50 μM ATP and a graded concentration of inhibitor at different concentrations of STAT5 peptide . Reaction mixes were incubated at 37 °C for 30 minutes. Next, an equal volume (25 μl) of ADP-Glo was added and plates were incubated at room temperature for 30 minutes, to deplete the remaining ATP and terminate the kinase reaction. Next, 50 μl of kinase detection reagent was added to each well to convert ADP to ATP that was measured by luciferase/luciferin reaction. Luminescence was measured 1 hour after adding the Kinase Detection Reagent during this time plates were incubated at room temperature. Luminescence correlates with the amount of ADP , and was recorded with GloMax 96 Microplate luminometer  and plotted with Prism software (Graphpad).Labelfree MicroScale Thermophoresis binding experiments were carried out with 40 or 1500 nM target protein in 25 mM Tris-HCl pH 7.5 + 150 mM NaCl + 0,1% Tween with varied concentrations of small compound at 40% MST power, 80% LED power in standard zero background capillaries on a Monolith NT.labelfree device at 25 °C . For fedratinib a ligand dependent quenching effect was detected. Due to this, the raw fluorescence was used for analysis. For the compound ruxolitinib the Thermoporesis + TJump was analyzed. The recorded fluorescence was normalized to fraction bound , processed using the software KaleidaGraph 4.5 and fitted using the Kd fit formula derived from the law of mass action. Technical duplicates were performed for each experimental setup.Fluorescence MicroScale Thermophoresis binding experiments were carried out with 5 nM Cy5-labeled target protein in 25 mM Tris-HCl pH 7.5 + 150 mM NaCl + 0,1% Tween with varied concentrations of small compound at 40% MST power, 20% LED power in hydrophilic capillaries on a Monolith NT.115 pico device at 25 °C . For fedratinib a ligand dependent quenching effect was detected. Due to this, the raw fluorescence was used for analysis. For the compound ruxolitinib the Thermoporesis + TJump was analyzed. The recorded fluorescence was normalized to fraction bound , processed using the software KaleidaGraph 4.5 and fitted using the Kd fit formula derived from the law of mass action. Technical duplicates were performed for each experimental setup.BaF3 cells expressing native JAK2-V617F and resistant variants Y931C and L983F were transduced with retrovirus co-expressing firefly luciferase and Cherry fluorescent protein. HEK293T cells were transfected with pMSCV-Luc-cherry-GW (firefly luciferase amplified from pLVX-luciferase by PCR and cloned in pMSCV-cherry-GW vector) to generate high titer viruses. For engraftment, 1 million cherry-positive cells were injected into tail veins of syngeneic female Balb/c mice . Starting 3 days later, mice were IP injected twice daily with 100 mg/kg of ruxolitinib. On days 7 and 14, mice were IP injected with luciferin (Promega) and imaged on an IVIS Spectrum imager ; images were analyzed using Living Image software. All experiments involving mice conformed to the National Institutes of Health Guide for the care and use of laboratory animals and were approved by the Cincinnati Children’s Hospital Institutional Animal Care and Use Committee (IACUC).Bone marrow mononuclear cells from PV patients were collected with informed consent. Institutional Review Board (IRB) approval was obtained from Cincinnati Children’s hospital or UCSF. The JAK2 cDNA was amplified using PCR. PCR products were sequenced and analyzed using the Lasergene Genomics Suite (DNASTAR) to identify any mutation.How to cite this article: Kesarwani, M. et al. Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance. Sci. Rep.5, 14538; doi: 10.1038/srep14538 (2015).


In parallel, the EIS field-effect structure allows the real-time electrochemical monitoring of the protein phosphorylation by detecting the release of protons associated with the kinases activity. This innovative combination of both field-effect and nanoplasmonic sensing makes the detection of protein phosphorylation more reliable and effective. As a result, the screening of protein kinase inhibitors becomes more rapid, sensitive, robust and cost-effective.Phosphorylation by kinases is an important post-translational modification of proteins. It is a critical control for the regulation of vital cellular activities, and its dysregulation is implicated in several diseases. A common drug discovery approach involves, therefore, time-consuming screenings of large libraries of candidate compounds to identify novel inhibitors of protein kinases. In this work, we propose a novel method that combines localized surface plasmon resonance (LSPR) and electrolyte insulator semiconductor (EIS)-based proton detection for the rapid identification of novel protein kinase inhibitors. In particular, the selective detection of thiophosphorylated proteins by LSPR is achieved by changing their resonance properties  ELISA or immunoblotting) are time-consuming. Radioisotope-labelling entails the use of expensive and hazardous reagents and is not easily available for all investigators. In recent times, some groups have described the development of sensitive and selective electrochemical1011121314in vitro and are suitable for screening of large libraries of candidate compounds has not been achieved yet.To regulate various cellular activities, proteins undergo post-translational modifications. These modifications cause conformational changes in the structure and activity of proteins Previous electrochemical methods attempted the detection of protein phosphorylation by measuring one of the following two chemical events: the addition of negative charges to the protein with the transfer of phosphoryl groups1717i.e. conjugated to a phosphate containing a sulfhydryl group replacing a hydroxyl group). Thio-phosphorylated proteins present the remarkable ability to bind to gold nanoparticles (AuNPs), which allows their electrochemical18This study reports for the first time a dual-mode sensor that uses an electrolyte–insulator–semiconductor (EIS) field-effect device coupled with nanoplasmonic effects measured by a localized surface plasmon resonance (LSPR) technique within the same experimental platform. During phosphorylation of proteins, the phosphate group at the γ-position of the adenosine triphosphate (ATP) is transferred to the serine, threonine or tyrosine amino acids of the proteinIn this study, the detection of protein kinase C alpha (PKC-α) activity is used as a model to demonstrate the applicability of the dual-mode AuNP-based LSPR and EIS sensors. PKC-α has been found implicated in several disease states, including cancer2224263N4 (Si-SiO2-Si3N4) wafer using 3-glycidoxypropyltrimethoxysilane (GOPTS) for 1 h at room temperature inside a customised teflon well; subsequently thio-phosphorylation of MBP by the PKC-α kinase was carried out in the presence of ATP-S. Three sets of control reactions were performed: one without PKC lipid activator; a second with the GF109203X inhibitor added before putting the kinase activator; and a third reaction where the protein phosphorylation was carried with ATP instead of ATP-S. Upon phosphorylation of MBP in the presence of ATP-S, the protein becomes thio-phosphorylated. The thio-phosphorylated proteins were then exposed to AuNPs (16 nm diameter average size), wherein the affinity between the thiol groups and Au resulted in the attachment of AuNPs to the thio-phosphorylated substrates. Generally, the covalent attachment of quasi-spherical AuNPs to the thiol groups occurs at a fast rate so after 10 min the substrates were rigorously washed with the reaction buffer to remove electrostatically attached AuNPs. As a consequence, the phosphorylated protein was labelled with AuNPs that enabled its detection through LSPR, alongside pH changes on the EIS substrate.The schematic of the phosphorylation reaction in our experimental conditions is presented in i.e. it is a reliable measurement of PKC-α activity). The reaction where ATP-S was replaced by ATP  showed around 9 nm of average wavelength shift, thus confirming non-specific binding of AuNPs to MBP (i.e. independent of thio-phosphorylation). This phenomena was however significantly lower than the true positive signals but did not affect the measurements. When the reaction was performed in the presence of a known inhibitor of kinase, GF109203X, a 7 nm of shift in wavelength was observed. These results support the hypothesis that inhibitors could be screened using the LSPR approach.As shown in 3N4, protein thio-phosphorylation could also be followed electrochemically as we previously describedvs 5 mV at the same time point). This result is consistent with previous reports28As the chemical reaction was performed on Sii.e. no kinase activator) and phosphorylation in the presence of PKC inhibitor are shown in The experiments shown in Interestingly, the variation observed in the LSPR shifts from sample to sample was larger in the case of thio-phosphorylated samples than to the EIS signal for the same samples. The variation in the LSPR signals can be attributed to: 1) inconsistency in the shape and size of the nanoparticles; 2) non-homogeneity of the nanoparticles; 3) variable density of AuNPs from sample to sample to which LSPR signal is sensitive. TEM analysis of the thio-phosphorylated substrates confirmed the irregularity in the shape and size of the nanoparticles in addition to variable density and heterogeneity among individual AuNPs in form of tetrahedral, cubic, octahedral and hexagonal phases that was apparent from the scans . The spa3N4 samples were silanized with 3-mercaptopropyl triethoxysilane (MPTES), which provides a free thiol group on the surface for direct AuNP binding. A full monolayer of AuNPs (or AuNPs aggregates) is expected with this surface chemistry. Upon immobilization of AuNPs, LSPR shifts of 19.3 ± 1.6 nm, 23.3 ± 2.2 nm and 31.3 ± 2.5 nm were observed for AuNPs of diameter 5 nm, 10 nm and 20 nm, respectively. These results indicate that the AuNP heterogeneity observed in Since LSPR shits depend on the size of AuNPsThe physical dimensions of the AuNPs are much larger than those of MBP. It is therefore possible that a similar number of AuNPs is attached for different amounts of thio-phosphorylation. In order to test for this possibility, the density of proteins immobilized on the surface can be controlled and checked for thio-phosphorylation with LSPR. To control the distribution of proteins on the surface, self-assembled monolayers (SAMs) of APTES/AHS  with 0.01%, 0.1%, 1%, 10%, and 100% of concentration of APTES were tested. This leads to coatings with different density and protein distribution that can be used to test the effect of protein density distribution on thio-phosphorylation detection using our EIS/LSPR technology. In order to check the effectiveness of this new method of controlling protein immobilization by using different solution ratios of APTES/AHS, horse radish peroxidase (HRP) was used as a test protein. Initial experiments were performed by immobilization of HRP with varied concentrations of APTES and testing the levels of HRP using a TMB  assay. . Higher i.e. on-off response, possibly suggesting saturation of binding sites for AuNPs at low thio-phosphorylation levels). To validate the sensitivity of LSPR for the identification of kinase inhibitors at this concentration of kinase, thio-phosphorylation experiments were conducted in the presence of PKC inhibitor (0.1 μM) – in this case both the LSPR and EIS signals are at their minimum  in reflection mode, which confirmed the presence of strong sulphide bonds in the thio-phosphorylated samples. Strong peaks of sulphide were observed at 1090 nm where the sample was phosphorylated in presence of ATP-S  for high throughput analyses of protein kinase activity. As a result, the screening of protein kinase inhibitors becomes more rapid, sensitive, robust and cost-effective. This is paramount for drug discovery in pharmaceutical industries and academia.This study describes a new method for detecting phosphorylation of proteins. Owing to the unique optical properties of AuNPs, a simple LSPR-based technique coupled to a field-effect device was developed for the evaluation of kinase activity and inhibition. LSPR gives a ‘on-off' response upon thio-phosphorylation; even at low concentrations of phosphorylation where EIS gives a small positive response, LSPR can be used to validate it as a true signal. Our approach demonstrates that multiple sensing techniques can be employed on the same sensing platform to achieve high levels of sensitivity and accuracy in the measurements. We also propose a novel method for controlling immobilisation of proteins via silanization by changing solution ratios of APTES/AHS. The combined detection method that we propose can easily be extended to multiplexed approaches , PKC-α kinase inhibitor GF 109203X, adenosine tri-phosphate (ATP), adenosine 5'-[γ-thio] triphosphate (ATP-S), 3-mercaptopropyl triethoxysilane (MPTES), 3,3′,5,5′-tetramentylbenzidine (TMB), allyl hydroxyl silane (AHS) and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich. Dephosphorylated myelin basic protein (MBP), purified from bovine brain using FPLC liquid chromatography, was purchased from Millipore. Protein Kinase C alpha (PKC-α) human recombinant kinase produced in Sf9, was procured from ProSec-Tray TechnoGene Ltd. PKC lipid activator cocktail was obtained from Millipore. Polyclonal phospho-(Ser) PKC substrate antibody was purchased from Cell Signaling Technology. 20 nm research grade gold nanoparticles (AuNPs) were bought in colloidal form from Diagnostic Consulting Network.All chemicals were of analytical grade and were used as received, unless otherwise specified. All aqueous solutions were made with double de-ionised water, 18.2 MΩ cm, with a Pyrogard filter . Tris base, MgCl3N4 samples were prepared in the same way as described in a previous report3N4 was deposited by plasma-enhanced chemical vapour deposition (PECVD) onto 4-inch n-type Si wafers with 50 nm of SiO2. The Si-SiO2-Si3N4 wafer was cleaned using a standard RCA wafer cleaning protocol. After cleaning, 100 nm aluminium was physically deposited on the back of the Si wafer to serve as an ohmic back-contact using an Edwards e-beam evaporator. The Si3N4 surface was then cleaned using acetone vapour at 110°C. For the electrolyte–insulator–semiconductor measurements, the wafer was sandwiched between a teflon well with an o-ring and a conductive plate (copper), so that the aluminium coated side of the wafer sits on the lower conductive plate. This formed a planar Si3N4 well with 19.64 mm2 interrogation area for the reaction defined by the size of the o-ring (5 mm diameter).The Si3N4 as described in a previous report2). The whole reaction volume was fixed to 100 μl for all replicates and controls. 1 μM ATP-S and 4 units of PKC-α (40 mU/μl)) were subsequently added. To initiate the phosphorylation reaction, PKC lipid activator (1:20 of reaction volume) containing 0.5 mg/ml phosphatidylserine and 0.05 mg/ml diacyglycerol in 20 mM MOPS (pH 7.2), 25 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothretiol and 1 mM CaCl2, was added. Three sets of control reactions were performed, one without PKC lipid activator, second with 0.1 μM PKC kinase inhibitor (GF 109203X), that were added before adding the kinase activator and the third reaction where protein phosphorylation was carried with ATP instead of ATP-S. 30 minutes after the start of the reaction, AuNPs were added and later the substrates were rinsed rigorously with reaction buffer to remove unbound gold nanoparticles.The protein was immobilized on the Sivia the SpectraSuite software (cross-platform spectroscopy operating software from Ocean Optics). Transmission electron microscopy (TEM) was done at 200,000× with JEOL JEM1200EXII transmission electron microscope with high contrast pole pieces. For TEM, 300-mesh nickel grids, coated with a fine layer of carbon, were used as substrates for the protein fractions in the microscope. MBP was immobilized in the same way as described in the above sections. The Fourier Transform Infrared Spectroscopy (FTIR) was done directly over the surface of Si3N4 using a Perkin Elmer Frontier FTIR instrument in reflection mode and a high resolution Mercury Cadmium Telluride (MCT) detector.The instruments for LSPR namely, reflection probe (R400-7UV-VIS), halogen light source (LS-1-LL) and the spectroscope (USB4000-UV-VIS-ES) were purchased from Ocean Optics. Before taking any signal from the scope, the system was calibrated for dark and light spectrum modes. The LSPR signal was then recorded in absorption mode by observing the wavelength dependence of the light absorbed through by nanoparticles C–V) characteristics of the Si3N4/SiO2/Si EIS structure. C–V measurements were performed using a CompactStat digital potentiostat . A conventional three-electrode electrochemical setup was employed with an Ag/AgCl reference electrode immersed in the electrolyte via a salt bridge used to apply the gate voltage and a Pt counter electrode. During the measurements, the gate voltage  was varied between −2 and +4 V, superimposed with a small ac signal of 10 mV at 1 kHz. The first measurement for the reaction was taken after adding ATP and kinase, i.e. before the start of phosphorylation process. After adding the kinase activator, the activity of the reaction was studied by recording the C–V characteristics every 2 minutes for 10 minutes. Finally the measurements were taken at 20, 40 and 50 minutes after the start of phosphorylation. In control reactions, C–V measurements were taken at the same time intervals. Each experiment was repeated at least three times and the reported data correspond to the average values.PKC-α activity was measured by analysing Capacitance–Voltage (N.B. conducted the experiments, prepared all figures and wrote the main part of the manuscript text. M.D.L., G.P. and P.E. supervised different aspects of the work and contributed to the writing of the manuscript text. All authors reviewed the manuscript.Supplementary Information


Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.Establishing the  In these studies, changes in the energy transfer between FRET-acceptor and FRET-donor moieties, engineered within the receptor sequence, provide a read-out of changes in receptor conformation on agonist binding –10. Alte–artments . These aartments –10 or a rtments –, and theAn alternative approach considered here is to monitor the phosphorylation status of GPCRs as a read-out of receptor activation. This is based on the “classical” principle that the conformation adopted by a receptor upon agonist occupation reveals phosphorylation sites, often within the third intracellular loop and C-terminal tail, that otherwise are not accessible in the inactive receptor conformation . In this1-muscarinic acetylcholine receptor (M1 mAChR), which is one of five muscarinic receptor subtypes (M1–M5) that respond to the natural ligand acetylcholine and is a subtype that has been implicated in a number of neurological processes (–228) in the third intracellular loop, which we show is a phosphorylation event highly sensitive to agonist stimulation. This antibody was used here to probe the phosphorylation status of the M1 mAChR following engagement with orthosteric and allosteric muscarinic ligands, both in vitro and in vivo. Furthermore, this antibody was used to determine that the M1 mAChR was activated in specific regions of the hippocampus following fear conditioning in a manner that maps to the regions of the hippocampus showing increased synaptic activity during memory acquisition. In this way we not only link the activation status of the M1 mAChR to memory acquisition but also establish the principle that phosphorylation sites can be used to probe the activation status of GPCRs during physiological responses and on drug treatment.We test this notion here by focusing on the Mrocesses , 14 mostrocesses –17. We hocesses –, althougUnless otherwise stated, all chemicals were purchased from Sigma. Xanomeline was obtained from Lilly .1 mAChR (CHO-M1 cells) were generated using the Flp-InTM system. CHO Flp-In cells were co-transfected with pcDNA5FRT containing M1 mAChR and pOG44, and transfected cells were selected with hygromycin B, and expression of M1 mAChR was confirmed by immunoblotting with anti-HA antibodies and by N-[3H]methylscopolamine (NMS) radioligand binding.Chinese hamster ovary (CHO) cells that stably and constitutively expressed the C-terminal HA epitope-tagged mouse Mm NaCl, 1.3 mm CaCl2, 4.3 mm KCl,1.17 MgSO4, 4.17 mm NaHCO3, 11.7 mm glucose, 10 mm HEPES (pH 7.4)) and incubated in this buffer containing 100 μCi/ml [32P]orthophosphate for 1 h at 37 °C. Cells were stimulated for 5 min with test compounds and immediately lysed by addition of buffer containing 20 mm Tris (pH 7.4), 150 mm NaCl, 3 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate. The M1 mAChR was immunoprecipitated from the cleared lysates using anti-HA affinity matrix (Roche Applied Science). The washed immunoprecipitates were separated by SDS-PAGE on 8% gels that were then dried, and radioactive bands were revealed using autoradiography film. The films were scanned and bands were quantified using AlphaImager software .Cells were plated in 6-well plates at 200,000 cells/well 24 h before experimentation. For phosphorylation experiments, cells were washed three times with Krebs/HEPES buffer without phosphate . After centrifugation at 20,000 × g, the resulting supernatant was diluted 1:1 with PBS, and the receptor was then purified on anti-HA affinity matrix. After extensive washing with solubilization buffer containing 0.5% Nonidet P-40, the resin was resuspended in 2× SDS-PAGE sample buffer. The sample was resolved by SDS-PAGE on 10% gels and stained with colloidal Coomassie Blue. The band associated with the M1 mAChRs was excised from the polyacrylamide and washed three times for 15 min with 100 mm triethylammonium bicarbonate (TEAB). Reduction and alkylation of cysteines were performed by addition of 10 mm dithiothreitol in 50 mm TEAB at 60 °C for 30 min followed by addition of 100 mm iodoacetamide in 50 mm TEAB for 30 min in the dark. Gel slices were washed three times for 5 min with 50 mm TEAB containing 50% acetonitrile, finally resuspended in TEAB containing 10% acetonitrile, and incubated overnight at 37 °C with 1 μg of sequencing grade trypsin . The resulting tryptic peptides were dried and resuspended in 1 ml of buffer containing 250 mm acetic acid and 30% acetonitrile, and phosphorylated peptides were enriched by addition of 20 μl of PHOS-SelectTM iron affinity resin and incubation at room temperature for 2 h with mixing. After washing the resin twice with loading buffer and once with water, tryptic phosphopeptides were eluted by addition of 200 μl of buffer containing 400 mm ammonium hydroxide and 30% acetonitrile. LC-MS/MS was carried out on each sample using an LTQ Orbitrap mass spectrometer . Peptides resulting from in-gel digestion were loaded at a high flow rate onto a reverse-phase trapping column  and eluted through a reverse phase capillary column (75-μm inner diameter × 150 mm) containing Symmetry C18 100-Å medium  that was self-packed using a high pressure packing device . The resulting spectra were searched against the UniProtKB/Swiss-Prot database using Mascot (Matrix Science Ltd.) software with peptide tolerance set to 5 ppm and the MS/MS tolerance set to 0.6 Da. Fixed modifications were set as carbamidomethylcysteine with variable modifications of phosphoserine, phosphothreonine, phosphotyrosine, and oxidized methionine. The enzyme was set to trypsin/Pro, and up to two missed cleavages were allowed. Peptides with a Mascot score greater than 20 and for which the probability (p) that the observed match was a random event was <0.05 were included in the analysis. The spectra of peptides reported as being phosphorylated were interrogated manually to confirm the precise sites of phosphorylation.Stably transfected CHO-M1 cells expressing mouse HA-tagged M1 mAChR. The 87-day program, which included four immunizations, was performed by Eurogentec. The resulting antisera were purified against the immunizing peptides. To generate antibodies for immunoprecipitation and detection of M1 AChR protein, rabbits and rats were immunized with the peptide RDRGGKGQKPRGKEQ that corresponds to amino acids 334–348 of the mouse M1 mAChR. The resulting antiserum was purified against the immunizing peptide.Phosphorylation-specific antibodies, anti-phosphoserine 228, 273, 322, and 451, were raised against peptide sequences AALQGpSETPGKG, RLLQAYpSWKEEE, KQPPKSpSPNTVK, and IPKRPGpSVHRTP corresponding to amino acid residues 223–234, 267–278, 316–327, and 445–456 of the mouse Mm HEPES, 110 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgSO4, 25 mm glucose, 58 mm sucrose (pH 7.4)) containing ∼0.3 nm [3H]NMS and increasing concentrations of acetylcholine in the presence of various concentrations of BQCA. Incubations were continued for 1 h at 37 °C. Nonspecific binding and filtration were carried out as above. Membrane-bound ligand was separated from free ligand by rapid filtration onto GF/B glass microfiber filters followed by three rapid washes with ice-cold 0.9% NaCl. Membrane bound radioactivity was determined by liquid scintillation  counting. Nonspecific binding was determined by the inclusion of atropine (1 μm) during the incubation with [3H]NMS.CHO-M1 cell membranes (50 μg/tube) were incubated in HEPES buffer . Membranes were prepared and solubilized in buffer composed of 20 mm Tris (pH 7.4), 150 mm NaCl, 3 mm EDTA, and 1% Nonidet P-40. The receptor was immunoprecipitated with the anti M1 mAChR polyclonal antibody. Immune complexes were washed three times in solubilization buffer and resuspended in 2× SDS-PAGE sample buffer. Receptors were separated by SDS-PAGE on 8% gels, transferred PVDF membranes, and immunoblotted with anti-phosphoserine 228 antibodies or antibodies against the M1 mAChR protein.To establish the presence and phosphorylation status of M1 AChR were seeded onto 20-mm glass coverslips for 24 h prior to experimentation. Cells were washed and incubated for 1 h in Krebs/HEPES buffer prior to treatment. Cells were fixed in PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min at room temperature. Anti-HA antibody was used at 50 ng/ml followed by Alexa FluorTM 546 goat anti rat secondary antibody at 1:1000. Anti-phospho-specific serine 228 antibodies were used at 0.25 μg/ml followed by goat anti-rabbit Alexa FluorTM 488 secondary antibody at 1:1000. Data were acquired using an Axiovert 200 M confocal laser scanning microscope (Zeiss).CHO-M1 cells expressing mouse HA-tagged Mmyo-[3H]inositol (PerkinElmer Life Sciences) for 24 h at 37 °C. Cells were washed twice in Krebs/HEPES buffer (pH 7.4) and incubated with 10 mm LiCl for 20 min at 37 °C. Appropriate concentrations of agonist were added for 20 min to stimulate [3H]inositol phosphate production. Where the allosteric interactions were studied, cells were pre-incubated with the allosteric modulator for 2 min prior to the addition of agonist. Incubations were terminated by aspiration of buffer and rapid addition of 500 μl of ice-cold trichloroacetic acid (TCA) (0.5 m). After extraction on ice for 20–30 min, samples were transferred to tubes containing 100 μl of EDTA (10 mm (pH 7.0)) and 500 μl of a 1:1 mixture of tri-n-octylamine, and 1,1,2-trichlorofluoroethane was added for 15 min at room temperature. After centrifugation at 20,000 × g for 2 min, 400 μl of the upper aqueous phase was transferred to fresh tubes containing 60 mm NaHCO3. [3H]inositol mono-, bis-, and trisphosphate ([3H]InsPx) fraction was recovered by anion-exchange chromatography on Dowex AG1-X8 formate columns. Columns were regenerated with 10 ml of ammonium formate (2 m)/formic acid (0.1 m) and washed thoroughly with distilled water. Samples were applied to the columns and the columns washed with 10 ml of distilled water. Columns were then washed with ammonium formate (60 mm)/sodium tetraborate (10 mm) solution. Total [3H]InsPx was eluted in 10 ml of ammonium formate (0.75 m)/formic acid (0.1 m) and collected in large scintillation vials. A 5-ml aliquot from the eluate was mixed with 10 ml of SafeFluor scintillation mixture, and radioactivity was detected by liquid scintillation counting.Cells seeded at 100,000 cells/well in 24-well plates were labeled with 2.5 μCi/ml 2) and transcardially perfused with 4% PFA. Following fixation, brains were immediately removed and further fixed overnight in 4% PFA.Male C57Bl6/NTAC mice (8–15 weeks old) were placed in the conditioning chamber (Stoelting ANY-maze fear conditioning system), and after a 2-min adaptation period, the mice received three tone/foot shock pairings, where a tone  always co-terminated with the foot shock . The conditioned stimulus-unconditioned stimulus pairings were separated by 1-min intervals. After completion of training, the mice remained in the conditioning chamber for 1 min and were returned to their home cages. For the immediate foot shock control, mice were put in the fear conditioning chamber, and a foot shock was immediately delivered ; 30 s latter, they were returned to their home cages. Mice remained in their home cages for 30 min before being anesthetized with 3% isoflurane (2 liters/min O1 AChR phospho-specific serine 228 at 2.5 μg/ml in blocking buffer (overnight at 4 °C). Sections were washed three times, and incubated with Alexa FluorTM 488 fluorescent secondary antibodies for 1 h at room temperature in blocking buffer. Following three washes, slices were mounted in Vectashield HardSetTM mounting medium with DAPI. All images were taken using a Zeiss confocal microscope with Zen software (Zeiss).Brains were processed in paraffin wax and sliced at 5 μm using a microtome. Following antigen retrieval, sections were washed in TBS containing 0.1% Triton X-100 and blocked for 2 h at room temperature in TBS, 0.1% Triton X-100, 10% goat serum, and 5% BSA. Sections were incubated with antibodies to c-Fos (Santa Cruz Biotechnology), ARC (Santa Cruz Biotechnology), and MTM plate reader.Extracellular signal-regulated protein kinase 1/2 phosphorylation assay in CHO-M1 cells were seeded into transparent 96-well plates at 35,000 cells/well and grown overnight at 37 °C. Cells were washed with PBS and incubated in serum-free α-minimal essential medium at 37 °C for at least 4 h. Cells were incubated with varying concentrations of agonist in the presence and absence of increasing concentrations of an allosteric compound for 5 min (compounds were co-added). The reaction was terminated by the removal of compounds and addition of 50 μl of SureFire lysis buffer. The lysates were agitated for 30 min at room temperature, and 4 μl of each lysate was transferred into a 384-well opaque Optiplate. SureFire detection mix (7 μl/well) consisting of detection reagent, activation reagent, donor beads, and acceptor beads (660:110:11:11 v/v) were added to the plate. Plates were incubated in the dark at room temperature for 2 h with gentle agitation before the fluorescence signal was measured using a PheraStar1 mAChR, were generated by genOway . Briefly, humanizing mutations V5A, S254T, K320R, G337A, and V413I were introduced into the mouse coding sequence to make the corresponding amino acid sequence identical to the human M1 mAChR. In addition, two point mutations, Y106A and A195G, were introduced that render the M1 mAChR insensitive to acetylcholine and promote sensitivity to the synthetic compound clozapine-N-oxide (CNO). This receptor mutant was termed the M1 DREADD receptor. Finally, an HA tag sequence YPYDVPDYA was appended to the C terminus followed by a stop codon. The gene-targeting construct consisted of long and short homology arms. The long arm consisted of a 5268-bp fragment containing the 3′ part of intron 2; this was generated by PCR from genomic DNA template using primers 85426GAlm 5′ GGGGACAACTTTGTATAGAAAAGTTGTTGGGAGGATGCAGCCTCTCCA and 85427GAlm 5′ GGGGACTGCTTTTTTGTACAAACTTGGGCGCGCCACGTACGCCTCTTCAAGGCTTAAGTGGAATGAAGGGGCAGCCCC. The short arm consisted of a 3188-bp fragment containing the 3′UTR sequence of exon 3 that was amplified from genomic DNA using primers 85424GAsm 5′ ACTGTCAACCCCATGTGCTACGCAC and 85425GAsm 5′ GGGCTACAAGGGAGCATGAACAAGC. A diphtheria toxin negative selection cassette was included to allow for selection of ES cells that had undergone homologous recombination, and a positive selection neomycin gene and a STOP of transcription cassette flanked by loxP sites were inserted upstream of the CHRM1 coding sequence. This construct was transfected into C57BL6 ES cells that were selected using neomycin. Resistant colonies were screened to verify correct integration of the 5′ and 3′ homology arms by PCR using primers 85488 5′ GCAGGTCGAGGGACCTAATAACTTCG and 85489sa 5′ AAGCTCTCAAGGTCCAACAGTTTCTGG for short arm integration and primers 85487la 5′ AGTCTTACCAGGGAAGAAAGCCTGATCC and 0070-Neo-16219sa 5′ CCTGCTCTTTACTGAAGGCTCTTTACTATTGC for long arm integration.Transgenic C57BL6/J mice, which express a humanized, mutated form of the MPositive clones were expanded and injected into blastocysts derived from C57BL6/J female mice to generate chimeric animals that were then bred with C57BL6 wild-type C57BL6 mice to generate heterozygous F1 animals. The neomycin/STOP cassette was excised by breeding heterozygous animals with Cre deleter mice to generate M1 DREADD constitutive knock-in mice. Genotyping was performed by PCR on genomic DNA obtained from tail biopsies using primers mM1 451 forward 5′ TTG GTT TCC TTC GTT CTC TGG GC and mM1 1227 reverse 5′ GAC GTA GCA AAG CCA GTA GCC CAG C. The resulting PCR products were subjected to restriction digestion with SacI, the products of which discriminated between WT C57BL6 and heterozygous and homozygous M1 DREADD animals.1 mAChR was conducted on Chinese hamster ovary cells expressing the mouse M1 mAChR (CHO-M1 cells) stimulated with the natural ligand acetylcholine or the M1/M4-preferring agonist xanomeline (A) (A).Our initial evaluation of the phosphorylation status of the Mnomeline A  A.1 mAChRs (B). To establish the precise sites of phosphorylation, a mass spectrometry-based phosphoproteomic study was conducted that identified nine serine and three threonine phosphorylation sites in the third intracellular loop of the receptor, as well as two serine phosphorylation sites in the C-terminal tail (C–F). Four of these sites at serine residues 228, 273, 322, and 451  were selected for the generation of phospho-specific antibodies based on the antigenicity of the sites.In phosphorylation studies, where total receptor phosphorylation was determined using metabolically labeled CHO-M1 cells, both muscarinic agonists increased the phosphorylation status of M1 mAChRs B. To estnal tail , C–F. Fo1 mAChRs from CHO-M1 cells (using antibodies that recognized the HA epitope engineered at the C terminus of the receptor) followed by treatment of the immunoprecipitate with calf intestinal alkaline phosphatase (CIAP) to remove the phosphate groups (A). Control immunoprecipitates were treated with vehicle. The immunoprecipitates were then probed in Western blots with the phospho-specific antibodies. In these experiments, treatment with CIAP removed the immunoreactivity for the M1 mAChR of all four phospho-specific antibodies (B) confirming that the antibodies were all phospho-specific.The phospho-specific nature of the antibodies generated was tested by immunoprecipitation of Me groups A. Controtibodies B confirm228, Ser273, Ser322, and Ser451 was regulated by agonist stimulation. Probing lysates derived from CHO-M1 cells stimulated with vehicle, acetylcholine, or xanomeline in Western blots using the phospho-specific antibodies revealed that there were low levels of basal phosphorylation on residues Ser228 and Ser273 but that agonist treatment significantly increased phosphorylation at these residues  could be established by monitoring the phosphorylation status of the receptor at Ser228. It is now well understood that the active conformation of GPCRs can also be promoted by ligands that bind at allosteric sites (A) . The enhanced binding of acetylcholine observed in the presence of BQCA was correlated with an augmentation of acetylcholine signaling as demonstrated by a leftward shift in the InsPx concentration-response curve to acetylcholine when conducted in the presence of BQCA . Hence, BQCA was seen to promote the active conformation of the M1 mAChR, which was reflected in the ability of BQCA to augment agonist binding and agonist-mediated signaling.The above data demonstrated that the activated state of the Mic sites A  conformation, we reasoned that BQCA would promote acetylcholine-mediated phosphorylation at Ser228. This was indeed the case, with BQCA mediating an ∼100-fold shift in the potency of acetylcholine-mediated phosphorylation at Ser228 (pEC50 (−log M) for acetylcholine = 7.5 ± 0.1 and pEC50 for acetylcholine in the presence of BQCA (3 μm) = 8.9 ± 0.3; Fig. 4E). Thus, phosphorylation at Ser228 not only reflected the action of orthosteric ligands at the M1 mAChR , but rather it acted to enhance acetylcholine responses (i.e. a concentration where BQCA acted as a PAM). However, at higher concentrations, BQCA could be seen to act as an agonist in its own right . Thus, high concentrations of BQCA in a pERK1/2 assay showed a direct agonist action (D). This agonist activity was reflected in complex concentration-response curves, where increasing concentrations of BQCA in the presence of very low concentrations of acetylcholine revealed the agonist activity of BQCA , whereas higher concentrations of acetylcholine revealed mixed agonist and PAM activities of BQCA . Importantly, when examined in phosphorylation studies, it was seen that at a high concentration (100 μm) BQCA mediated phosphorylation at Ser228 to a similar extent as that seen with a maximal concentration of acetylcholine (500 μm) . In contrast, a low concentration of BQCA (3 μm) alone did not stimulate any change in receptor phosphorylation .These phosphorylation experiments were conducted with BQCA at a concentration that in itself gave no response . However, following agonist treatment  or treatment with a high concentration of BQCA (100 μm), punctate cytoplasmic staining was detected (G). Interestingly, the punctate localization of the phosphorylated M1 mAChR represented only a small proportion of the total receptor population (identified using the antibodies against the HA epitope tag) that, after a 5-min stimulation, appeared concentrated at or very close to the plasma membrane (G).Phosphorylation at Sernditions G. Howevedetected G. Interemembrane G.1 mAChR on Ser228. This includes orthosteric ligands, such as acetylcholine and xanomeline, as well as allosteric ligands (i.e. BQCA). In this sense, Ser228 phosphorylation is an ideal sensor of receptor activation, because this phosphorylation event reflects activation of the receptor regardless of the mechanism of activation.By combining all these data, it can be concluded that muscarinic ligands that promote the transition of the receptor from an inactive R-state to an active R*-state promote phosphorylation of the M228 on the M1 mAChR can act as a biosensor for receptor activation in response to either orthosteric or allosteric ligands. To test whether this could be used to monitor the activation status of the M1 mAChR in vivo, we first employed a novel mouse model where the wild-type M1 mAChR gene was humanized so that the translated amino acid sequence was identical to the human M1 mAChR and where an HA epitope tag was inserted at the C terminus. Two further point mutations were introduced into the orthosteric binding site (Y106A and A195G) (A), which resulted in an HA-tagged humanized M1 mAChR mutant that was unable to be activated by the natural ligand acetylcholine but rather could be activated by the synthetic drug CNO (in vitro and termed as a M1 DREADD (1 mAChR promoter ensuring that the M1 DREADD would be expressed in the same cell types and at the same expression levels as the native M1 mAChR.The data presented above support the notion that phosphorylation of Serd A195G) A, which drug CNO , 27. Thi1 DREADD . The des228 (B). In contrast, the M1 DREADD was phosphorylated by CNO in a concentration-dependent manner (B). Immunohistochemical labeling of the HA epitope tag identified expression of the M1 DREADD mutant in the molecular layer of the CA1 region of the hippocampus (C). Using the Ser228 phospho-specific antibody in M1 DREADD identified light immunohistochemical staining in the CA1 region of the hippocampus that was up-regulated within 30 min of intraperitoneal administration of CNO (D).Analysis of the ability of acetylcholine and CNO to induce phosphorylation of the M1 DREADD was tested in transfected cells. It was found that acetylcholine had no significant effect on M1 DREADD phosphorylation at Ser228 B. In cont manner B. Immunopocampus C. Using n of CNO D.228 phospho-specific antibody revealed neurons where the M1-DREADD receptor existed in a phosphorylated state . Interestingly, not all of the M1-DREADD-expressing neurons stained with the phospho-specific Ser228 antibody suggesting that in some neurons the M1 receptor remained non-phosphorylated (at least at this residue) . It should be noted that M1-KO controls also revealed some weak nonspecific nuclear staining with the Ser228 phospho-specific antibody (D and E).Co-staining with anti-HA, to reveal the expression of the M1-DREADD HA-tagged receptor, and the Ser228 phospho-specific antibody can be used to monitor M1 mAChR activation following stimulation by a synthetic ligand. We therefore tested this further by the administration of xanomeline (5 mg/kg i.p.) into wild-type mice followed by immunohistochemical analysis of phosphorylation at Ser228. Pharmacokinetic analysis determined that xanomeline levels in the brain peaked 30 min after injection. At this time the animal was sacrificed by perfusion fixation, and the brain was removed and sectioned for immunohistochemical staining. This procedure revealed phosphorylation of the M1 mAChR at Ser228 in the neuronal cell bodies in the pyramidal layer of the CA1 region of the hippocampus (A). In contrast, low levels of staining were observed in vehicle-treated mice (A).The data in the M1 DREADD mice supported the notion that the Serpocampus A. In conted mice A.228 could also act as a biosensor for M1 mAChR activation in response to the administration of a PAM, BQCA was administered at 15 mg/kg, which resulted in a free concentration of BQCA in the hippocampus of 51.8 ± 3.6 nm; a concentration where BQCA would show PAM activity at the M1 mAChR. In these experiments, Western blots of hippocampal lysates prepared from BQCA-treated animals showed an increase in the phosphorylation status of the M1 mAChR at Ser228 in response to BQCA (B and C).To test whether phosphorylation at Ser to BQCA , B and C1 mAChR in the hippocampus could be monitored using the phosphorylation status of the M1 mAChR at Ser228, we next asked whether the phosphorylation of Ser228 could also be used as a biosensor for M1 mAChR activation during a physiological process. Previous studies had implicated a role for the M1 mAChR in learning and memory . Importantly, this increase in Ser228 phosphorylation correlated with an increase in c-Fos immunoreactivity, a marker of neuronal activity (D).Having demonstrated that the action of an orthosteric agonist and a PAM at the Md memory –17. Becaresponse . The mic control D. Importactivity D.1 mAChR can be used as a read-out for receptor activation. Using this antibody as a biosensor for the activated receptor, we show that the M1 mAChR is activated in the hippocampus following fear conditioning training. This correlates with an increase in neuronal activity in the hippocampus thereby strongly supporting a link between hippocampal M1 mAChR activity and hippocampus-based learning and memory  or might show different levels of phosphorylation in response to ligands. Such phosphorylation sites would not be suitable as indicators of receptor activation.It is important to point out that not all phosphorylation sites on GPCRs will show the characteristics of Ser228 antibody could be used to detect phosphorylation (and thereby activation) of the M1 mAChR in vivo, we employed a mutant mouse line that expressed a M1 DREADD receptor in place of the wild-type M1 mAChR. This receptor could be selectively activated by CNO in vivo  is exquisitely sensitive to agonist stimulation. Using this phosphorylation event as a biosensor for receptor activation, we have been able to establish the activation of the M1 mAChR in vivo in the CA1 region of the hippocampus following drug treatment and memory acquisition.In conclusion, by determining the phosphorylation sites within the MA. J. B. conducted the primary experiments and assisted in writing the paper. S. J. B., R. P., S. M. B., A. M., J. M. B., T. M. H., J. M. E., and A. R. B. contributed to the experimental data. R. A. J. C., L. M. B., and C. C. F. contributed to experimental design and data analysis. A. B. T. conceived and led the study and wrote the paper.


They are also involved in chromatin structure regulation, gene expression, intracellular signalling pathway modulation and development. All essential lamin functions seem to depend on their capacity for assembly or disassembly after the receipt of specific signals, and after specific, selective and precisely regulated interactions through their various domains. Reversible phosphorylation of lamins is crucial for their functions, so it is important to understand how lamin polymerization and interactions are modulated, and which sequences may undergo such modifications. This review combines experimental data with results of our  All the discovered lamins share the same overall domain structure independently of the organism of origin ,2. The sPolymerization of lamins is essential for their mechanical functions, and all the domains are involved in this process. The head and tail domains contain two conservative motifs called ‘mitotic’ phosphorylation sites (cdc2/Cdk1 sites) that are crucial for the regulation of nuclear lamina and lamin disassembly in most metazoans . Lamins Caenorhabditis elegans has only a single gene coding for a single Ce-lamin protein, which functionally resembles both A- and B-type lamins of vertebrates, although it lacks some of the typical structural features of vertebrate B-type lamins. In Drosophila melanogaster, two lamin genes are present, coding for lamin Dm (resembling B-type lamins) and lamin C (resembling A-type lamins) [In general, two major types of lamin are distinguished: A-type and B-type. The number of genes for lamins increased during the evolution of the metazoans.  lamins) –9.LMNA gene codes for lamins A (and AΔ10), C and the additional variant C2, which is mainly produced in the reproductive cells. The LMNB1 gene codes for lamin B1, and LMNB2 codes for lamins B2 and B3 [Mammals typically have three lamin genes, coding for lamins A/C, B1 and B2. As a result of alternative splicing, more than three protein products are synthesized. The  A and AΔ, C and tA more complex system of lamin genes and proteins exists in other vertebrates. In teleost fish, amphibians and birds, apart from the genes coding for lamins A (with no splicing to lamin C), B1 (LI) and B2 (LII), there is also a gene coding for lamin B3 (LIII), the expression of which is almost exclusive to the oocyte and early embryo. The lamin B3 gene in amphibians codes for three proteins due to alternative splicing: lamins B3a (LIIIa), B3b (LIIIb) and LIV . There iα [The unquestioned main role of lamins is their mechanical and structural function as major building blocks of the karyoskeleton. Lamins A and C have also been implicated in the regulation of chromatin structure, intracellular signalling pathways, stiffness, plasticity and development ,12. Reveα –16 (for α ), bindinα  and bindα . Specifiα ,21.As lamin functions depend on the capacity for assembly or disassembly and specific, precisely regulated interactions, it is crucial to understand which domain of the lamins is responsible for polymerization and binding partners, and how the processes are modulated. It is widely known that lamins undergo post-translational modifications via phosphorylation, farnesylation, myristoylation, ubiquitination, acetylation, sumoylation and proteolytic cleavage . The onlOur study focused on the conserved domains of particular lamins, their function and their biological effect depending on these reversible reactions. The frequency of discovered phosphorylation is significantly higher in the head and tail domain than in the rod domain, so we principally investigated these two regulatory domains.C. elegans, D. melanogaster, Danio rerio, Xenopus laevis, Gallus gallus, Mus musculus and Homo sapiens, and marked conservative, specific sequences, both within all the analysed lamins together , Ce-lamin does not have a typical Cdk1 site [In vitro studies demonstrated that this Cdk1 site is crucial, together with other sites, for phosphorylation-dependent polymerization and depolymerization of lamins.The head domain of lamins contains at least one Cdk1 cdc2) phosphorylation site motif, also called the lamin N-terminal mitotic site  ‘mitotic’ site in lamins is also predicted to be an efficient target for phosphorylation by other kinases, such as Cdks (Cdk1–6), glycogen synthetase kinase (Gsk)3 and extracellular signal-regulated kinase (ERK)1–2 . Experimal cells , and by al cells . Moreoveal cells .The similar, conservative motif in most lamins  also contains strong consensus sequences for Cdk5, Gsk3 and ERK1–2 kinases. Phosphorylation of this site has been discovered experimentally  breast cancer-associated protein 1 C-terminal repeat (BRCT) domain, and polo-like kinase 1 polo-box domain (Plk1 PBD) and 14–3–3 protein [All lamins have predicted  protein . METP an protein ,51. This protein  are repeEach lamin contains also at least one predicted 14–3–3 domain binding motif (RXXpS) . This moXenopus lamin A/B3 and B1/B2, respectively [D. melanogaster has two copies and lamin Dm has a single copy. This may reflect the adaptation of lamins to the biology of organisms in which oocytes are stored in the ovaries for long periods of time in prophase (diplotene). For example, in goldfish a major component of the oocyte nuclear lamina is lamin B3, and when Cdk1 kinase is not active, a still unknown SRPK kinase phosphorylates lamin B3 at the Cdk1 site [Moreover, fish and amphibian lamins B3 contain SR/RS dipeptide motifs specific for serine/arginine protein kinases (SRPKs). Such a motif is present in four copies in goldfish and torafugu, in three copies in zebrafish, and in one and two copies in ectively . This modk1 site ,55.An interesting sequence motif is located in fly lamin C (S13RASTSTP). It is similar to the B-type lamin C-terminal motif  impairs polymer formation [in vitro polymerized lamin network [It is known that the lamin head domain is necessary for head-to-tail lamin polymerization. The direct experimental evidence of the importance of the head domain of lamins comes from ormation . Bacteriormation . Moreove network . Also, h network .As the head domain is essential for head-to-tail polymer formation, phosphorylation of sites on the head domain would keep a fraction of lamins in an unpolymerized state  or may introduce gaps in lamin polymers facilitating lamin and lamin filament flexibility, mobility and redistribution within the nucleus. Additionally, many sites recognized by different kinases have been identified in the head domains of lamins, thus different signalling pathways may affect lamin polymerization through the head domain –63. This3.3.1.b, 3–5). Proximal to the end of the rod domain lies the C-terminal ‘mitotic’ cdc2 (Cdk1) site, crucial for lamin polymerization and depolymerization (with the exception of Ce-lamin). This region contains conservative phosphoacceptor serine residues, which in H. sapiens are located at positions S390 and S392 in lamin A/C [Xenopus, the predicted phosphoacceptor residues are S386/388, S391/393 and S395/397 for lamins A, B1 and B2, respectively. Phosphorylation of this region by Cdk1 was also confirmed experimentally for murine lamin A (S390/392) [The tail domain of lamins contains several conserved sequences essential for all lamins ,71  ,26 and f390/392) . Near th390/392) . The pho390/392) ,32,33,7290/392 ,2in vivo [This canonical, conserved sequence of the C-terminal ‘mitotic’ site is not present in worm Ce-lamin and fish lamin B3. Fly lamins (Dm and C) have different sites for Cdk1 phosphorylation, and these are predicted to be modified by an entire set of kinases  . In lamiin vivo  and by lin vivo . Pseudopin vivo .3.2.H. sapiens are located at positions S403/404 in lamin A/C [in vitro and in vivo [The next conserved motif in the lamin tail domain is the sequence denoted as S/TRAS/T b and 3. amin A/C , S405 inamin A/C ,32,33 anamin A/C . This reamin A/C . Phosphoamin A/C  and larg in vivo ,37. More in vivo .D. melanogaster, this sequence is predicted as a target for the entire set of kinases. Note that a similar motif in fly lamin C is also located in the head domain  but within a much less conserved sequence. This suggests differences in functions of this region between lamin types.The detailed analyses of sequences within particular lamin types revealed additional lamin-type specific features . A-type Also, B1 lamins have an additional stretch of negatively charged amino acids immediately after the NLS, while in lamin A and lamin B2/B3 this stretch is more variable. As this region was reported to contribute to DNA/histone/chromatin binding, this may account for the differences in chromatin- and DNA-binding affinity for lamin A and lamin B (see below for discussion).3.3.Ciona intestinalis (sea squirt) lacks it [in vivo [The next conserved globular region, over 105 amino acid residues in length, called the Ig fold , is resplacks it . The crylacks it ,75,76. N[in vivo .It is also believed that the Ig fold participates in chromatin and DNA binding together with the S/TRAS/T sequence, NLS signal and polar amino acid residue stretches surrounding these sequences. Available data support this assumption, as different properties of lamin A, progerin and B-type lamins with respect to chromatin binding were reported –80.Phosphorylation sites were also experimentally identified in the Ig fold, but with lower frequency. It has been reported using phosphorylation-specific antibodies that S458 in the Akt1 motif is specifically phosphorylated only in myopathy patients , but pho3.4.in vivo and is predicted to be a consensus sequence for casein kinase II —CaaX figures d and 5 ,58,81–83in vivo are sparse. In silico analyses of the available experimental data analysed against the structure of the active site pocket of farnesyl transferase suggest different efficiency of farnesylation of different sequences [in vivo. If this is true, these lamins may be more easily located inside the cell nucleus than other B-type lamins.Next to the CaaX motif, lamins have conserved proline (-5 in lamin A and -7 in B-type lamins), which sterically helps to accommodate the farnesylation motif into the farnesyl transferase active site pocket. Although all lamins have this farnesylation motif (CaaX), experimental data on the efficiency of farnesylation of particular sequences in lamins equences . This su3.5.Tailless lamins associate predominantly in parallel, forming dimers as building blocks. About 40 amino acid residues of the tail region  are required to form a predominantly longitudinal polymer ,59,88. T4.4.1.in vivo identified phosphosites in lamins other than human, so we used bioinformatics tools for phosphorylation site prediction. We used three programs utilizing different algorithms: NetPhos v. 3.1 [inasePhos v. 3.10 [Limited data are available from studies of particular lamins or large-scale phosphoproteome mapping on s v. 3.1  DISPHOS s v. 3.1  and Kina v. 3.10 . They we v. 3.10 .in vivo sites in human and fly lamins. This suggests that when no experimental data are available, this approach may be useful.Detailed analyses of all lamin sequences detected different patterns of phosphosite location between lamins . The most frequently predicted phosphoacceptor sites are for PKA and PKC. The less frequently predicted sites are for Gsk-3, ERK1–2, Cdk1–5 and MAPKK. Most of the consensus sites are localized within conserved sequences, which suggests similar mechanisms of lamin regulation between different lamins and different species. Ce-lamin has many predicted PKC and PKA sites, but does not have any predicted Cdk1–5 or ERK1–2 sites and only a few sites for GSK-3 kinases, which suggests less complex regulation mechanisms of biological function of this lamin in vivo .4.2.in vivo phosphorylation sites on lamins discovered during large-scale phosphoproteome mapping in various model systems. The biological samples under study are either from cell lines, normal tissues, organs, stimulated material or particular developmental stages. Although they may give us a lot of valuable data in general, they also may provide considerable false-positive data, so we graded the available records depending on how many times a particular phosphorylation site was identified, dividing the data into three groups: fewer than five datasets, 5–20 datasets and more than 20 datasets. That allowed us to indicate the most probable residues .Recently, large datasets have been made available on in vivo phosphorylation sites from large-scale phosphoproteome studies with individually mapped sites and illustrated them in diagrams showing common motifs in proteins. Almost all sites mapped in experiments focused on particular lamins were also confirmed via large-scale analysis. Note that the black frames in figures 2Data gathered from large proteome studies proved to be valuable, and may be used to indicate particular sequences and sites for precise studies of their function. Based on this assumption, we integrated available data on lamin 4.3.We have only very limited knowledge on the effect of specified phosphorylated residues in particular lamins. Although particular phosphosites were found in many biological samples, this often does not provide conclusions on the relationship with lamin properties or function, to what extent the lamin protein was phosphorylated, how many sites were modified simultaneously on a single molecule, or which kinases were involved.in vivo indicated different values ranging from 0.3 phosphates per molecule up to about three phosphates per lamin [Initial studies on lamin phosphorylation attempted to assess the number of phosphate moieties per statistical lamin. For instance, the data for lamin Dm er lamin ,46,92–94er lamin ,45,46. Aer lamin . Thus, ain vitro and in vivo [Furthermore, phosphorylation site motifs for particular kinases in lamins overlap with one another. This may lead to a situation where a particular phosphoacceptor amino acid residue in a lamin may be phosphorylated by an entire set of kinases. The best-known example of this effect is the N-terminal ‘mitotic’ consensus site and nearby sites, which can be modified not only by Cdk1, but also by other Cdks, Gsk3, ERK1–2/MAPK, PKA and PKC, and by virus kinases both  in vivo ,55,95–98in vitro, e.g. phosphorylation of (presumably) a ‘mitotic’ N-terminal phosphorylation site (S45) on lamin Dm by Cdk1 kinase depolymerized in vitro lamin Dm to the same extent as phosphorylation in vitro by PKC and PKA (presumably on S42 or S50) [Moreover, phosphorylation of a neighbouring site may have a similar effect on lamin properties  or S50) . These l5.5.1.in vitro and in vivo, and causes aberrant mitosis when mutated or pseudophosphorylated [Drosophila melanogaster lamin C is soluble upon single N-terminal Cdk1 site pseudophosphorylation (S37), while single C-terminal atypical Cdk1 site pseudophosphorylation in a tail fragment (T435) is sufficient for solubilization of lamin Dm [Phosphorylation of amino acid residues in the head and tail domain, especially those located next to the central coiled-coil domain, induces lamin depolymerization, might disrupt the nuclear lamina network orylated ,38,41,96orylated . It seemin vitro assembled lamin Dm can be triggered by Cdk1, as well as by PKA [Moreover, different effector kinases may trigger depolymerization of lamins during interphase and mitosis using a large variety of kinase motif sites , and lipid-mediated signalling takes part in this process ,98. For s by PKA  and humas by PKA . It is as by PKA . For exas by PKA . Interess by PKA .Another possible effect of lamin phosphorylation may be associated with mechanical properties of the nuclear lamina as a whole or locally. It is thought that regulation of plasticity and stiffness of the cell nucleus depends on A-type lamins and their interactions with other proteins (for review see ). Recentα, MAPK kinases and cofactors) or vice versa [These data confirm the importance of phosphorylation of lamins for location of regulatory proteins as proper phosphorylation of lamin A (and lamin C) may cause its relocation into nucleoplasm and position their interacting protein  domain ,123 and y system .It seems that the most important DNA/chromatin/histone-binding regions in B1-type lamins were mapped specifically to the sequences S/TRAT/S and NLS ,125,127.5.4.in vitro phosphorylation of chicken lamin B2 by PKC inhibits nuclear import [The mammalian lamin A sequence GRASS404  ar import . B-type Besides, phosphorylation of the chromatin-binding domain and the presence of farnesylation may also modulate the nuclear import of lamins. For example, only the double mutant of lamin Dm, T435E and the farnesylation defective, was fully cytoplasmic . Nuclear5.5.cis to trans conformation, inducing structural changes in the entire polypeptide chain [The S/TP motif present in lamins can be a target for both kinases and enzymes called peptidyl-prolyl isomerases (PPIs), which catalyse proline transition from de chain . Only onde chain .By changing the proline conformation, which may function as a ‘hinge’ site, Pin1 can enforce kinks into lamin polypeptide chains and influence lamin properties . Pin1 itself can also be switched off through phosphorylation (e.g. by PKA) and switched on by PP2A (protein phosphatase 2A) . RecentlDirect interaction of lamin and Pin1 was reported in the case of human fibroblast infection by cytomegalovirus . A smallThe S/TP motifs existing in lamins may function in different ways: as hinges for spontaneous or catalysed conformational changes and as part of a more complex system of regulation involving Pin1. They may keep their ‘fixed’ conformation after phosphorylation for a longer time than unphosphorylated ones. Thus, it is not phosphorylation alone, but phosphorylation in combination with active isomerases including Pin1 that may more precisely regulate lamin properties through S/TP phosphosites.6.The phosphorylation of lamins seems to be a major regulator of the properties of nuclear lamina. It is a crucial mechanism influencing lamin polymerization and interaction with other proteins. As there is still limited knowledge about the specific function of particular phosphosites, we attempted to indicate residues responsible for various functions and showed their importance connected with sequence conservation and the frequency of particular residue phosphorylation in different organisms and cell types. Based on the demonstrated conservation of domains with phosphosites between lamins, it is possible to draw predictions about the functions of domains and possible phosphosites in lamins that have never been tested.in vitro and in vivo can be observed. Different regulatory mechanisms and different sets of kinases acting in concert are involved in the regulation of lamin polymerization, although prolonged action of single kinases or cascades  may give the same effect. Potential kinases that may phosphorylate residues in consensus motifs for experimentally confirmed phosphosites were also pointed out. Their identification through in vitro or in vivo studies may reveal the plethora of signalling pathways involved in the control and modulation of lamin properties and functions.The plasticity of signalling networks regulating lamin polymerization, solubility, chromatin binding, location and interactions through phosphorylation both in vivo or chromatin bound versus unbound).The very intriguing aspect of lamin phosphorylation is the level of such modification in a single molecule. Lamins are hyperphosphorylated during mitosis, but the exact number of modified residues is usually not known. Very little is known about number of phosphates during interphase, despite many of them having been found. Older data suggest that phosphorylation is rather rare, that there are 0.3–3 phosphate groups per lamin molecule during interphase, and that lamins are hyperphosphorylated during mitosis. It was recently demonstrated that phosphorylation on so-called ‘mitotic sites’ increases several-fold during mitosis ,135. We in vivo. The major task of future studies in the field is to resolve the function of particular phosphosites or combinations of sites with respect to particular lamin functions and properties. We believe that a systemic in vitro and in vivo approach using pseudophosphorylation mutants and dominant negative mutants of lamins in studies directed at particular properties of lamins will prove most successful.Although studies on the functions of lamin phosphorylation have been conducted over the course of 35 years and a lot of data have been gathered on the location of phosphorylation sites in many model organisms, we are still far away from direct knowledge of the molecular mechanisms underlying the functions of particular sites 7.7.1.Caenorhabditis elegans; gi:17506429; ref:NP_492371.1LMN_C Lamin, Drosophila melanogaster; gi:442623692; ref:NP_001260974.1LC_D Lamin C, Drosophila melanogaster; gi:17136290; ref:NP_476616.1LDm_D Lamin Dm, Danio rerio; gi:190337691; gb:AAI63807.1LA_F Lamin A, Danio rerio; gi:40254675; ref:NP_694504.2LB1_F Lamin B1, Danio rerio; gi:366392938; ref:NP_571077.2LB2-F Lamin B2, Danio rerio; gi:42476244; ref:NP_694505.2LL3_F Lamin L3, Xenopus laevis; gi:156119433; ref:NP_001095210.1LA-X Lamin A, Xenopus laevis; gi:147904084; ref:NP_001080053.1LB1_X Lamin B1, Xenopus laevis; gi:147901703; ref:NP_001080947.1LB2_X Lamin B2, Xenopus laevis; gi:148236667; ref:NP_001081545.1LL3_X Lamin L3, Gallus gallus; gi:45384214; ref:NP_990618.1LA_G Lamin A, Gallus gallus; gi:45384220; ref:NP_990617.1LB1_G Lamin B1, Gallus gallus; gi:45384202; ref:NP_990616.1LB2_G Lamin B2, Mus musculus; gi:112378771; gb:ABI16251.1LA_M Lamin A, Mus musculus; gi:112378773; gb:ABI16252.1LC_M Lamin C, Mus musculus; gi:188219589; ref:NP_034851.2LB1_M Lamin B1, Mus musculus; gi:113195686; ref:NP_034852.2LB2_M Lamin B2, Mus musculus; gi:220472; dbj:BAA02708.1LB3_M Lamin B3, Homo sapiens; gi:27436946; ref:NP_733821.1LA_H Lamin A, Homo sapiens; gi:5031875; ref:NP_005563.1LC_H Lamin C, Homo sapiens; gi:5031877; ref:NP_005564.1LB1_H Lamin B1, Homo sapiens; gi:388240801;ref:NP_116126.3LB2_H Lamin B2, Protein sequences were obtained from the NCBI database .M. musculus was not aligned to the other lamins because its sequence is identical to the sequence of lamin B2 from M. musculus in the fragments that are presented in the figures. For human lamin B2, the full sequence (with 20 ‘new’ amino acids) was used; in previous numbering M21 was M1.Lamin B3 from 7.2.lustalX v. 2.0, using the BLOSUM matrix and the Gonnet matrix. Each alignment was edited in Jalview v. 2.8. and individually corrected for inaccurate fragments. For each particular alignment, data from each matrix and available experimental data were taken into account. Black frames mark particular phosphosites identified in large-scale proteomics studies only, while pink frames mark sites mapped both individually in lamin-specific experiments and in large-scale proteomic studies. There are some exceptions, also marked by pink frames, that were not confirmed by mass spectrometry: S5, T416, T480, S525 in murine lamin A and S19, S50 in fly lamin Dm . Motifs for only experimentally confirmed kinases are shown in the pictures.Sequences were aligned with CetPhos v. 2.0 server (http://www.cbs.dtu.dk/services/NetPhos), DISPHOS v. 1.3 (http://www.dabi.temple.edu/disphos/) and KinasePhos v. 2.0 (http://kinasephos.mbc.nctu.edu.tw/). NetPhos uses a method based on the neural network for predicting potential phosphorylation sites in protein sequences [inasePhos is based on the identification of protein kinase-specific phosphorylation sites, using the known phosphorylation sites, categorized by substrate sequences and their corresponding protein kinase classes [www.dabi.temple.edu/disphos). The program GenSite was used to present the phosphorylation sites in lamins  and KinasePhos v. 2.0.To predict specific motifs recognized by kinases, two programs were used: P