It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV–vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the structural diversity of the isozymes. These findings may represent additional mechanisms for the differential inhibitory effects arising from the coincubated AuNPs on the metabolic activities of the hepatic CYP isozymes.Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under  It is now well established that, in biological fluids such as blood, the surface of nanoparticles becomes coated with a complex mixture of proteins to form the so-called ‘protein corona,’ which affects the NP-protein interactions and eventually determines their fate in the body. Extensive studies have been carried out on the NP-induced protein corona phenomenon, and the intrinsic characteristic of NPs  and the complex non-covalent forces occurring at the nano-bio interface  are generally considered as the main factors controlling the surface adsorption and interactions -1) were investigated; the plasmonic optical adsorption, hydrodynamic diameter, and Z-potential of AuNPs were read at regular intervals.The same volume of 7- or 70-nm AuNPs and HLM were incubated for an hour. NPs in two concentrations  and HLM in five concentrations  using quartz cuvettes with an optical path length of 0.5 cm at room temperature. Several metallic NPs, such as gold and silver, exhibit a characteristic absorbance maximum in visible range, namely surface plasmon resonance (SPR). This characteristic wavelength changes depending on size and surface alterations. SPR is sensitive to the surroundings of NPs at a molecular level; therefore, the changes of NPs in a close environment (such as biomolecular corona) can be investigated using this technique.Dynamic light scattering (DLS) is a powerful tool for determining small changes in the size of NPs. The DLS and Z-potential experiments were performed on a Malvern Zetasizer Nano ZS . Samples filtered followed by equilibration  in a quartz cuvette to 37°C. The software was arranged with the specific parameters of refractive index and absorption coefficient of the material and the viscosity of the solvent. DLS allows determination of hydrodynamic diameter of colloidal particles and conjugates, that is the diameter of sphere with the same Brownian motion as the analyzed particle.To minimize the AuNP-associated matrix effects on liquid chromatography-mass spectrometric measurement of the enzymatic activities, we developed a novel nanoprecipitation approach for pretreating samples from the complex coincubation mixture. In a previous study, we have reported a facile salt-triggered precipitation method to efficiently remove water-soluble nanoparticles and extract analytes from complex NP-containing bio-samples into an organic phase for direct liquid chromatographic analysis . In thatAs shown in Table Km substrate concentration and different microsomal protein concentrations. AuNPs show no obvious effects on CYP450s at low particle concentration, but if the concentration is increased, CYP inhibition is observed  with the lowest IC50 value (61.04 μM), followed by testosterone 6β-hydroxylation (3A4), tolbutamide methylhydroxylation (2C9), (s)-mephenytoin 4′-hydroxylation (2C19), and felodipine dehydrogenation (3A4), with the corresponding IC50 values of 68.3, 103.4, 137.1, and 203.9 μM, respectively. However, at the same mass concentration, AuNPs with different sizes (5 to 70 nm) showed different effects on CYPs. Smaller particles, particularly 5-nm AuNPs, showed pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4, while 100-nm AuNPs caused minimal effect on these six CYP450-selective activities. The smallest effect was observed on phenacetin O-deethylase (1A2), even 5 nm at concentration above 300 μM caused a less than 10% inhibition of this enzyme. The result accords closely with earlier reports, where CYP1A2 was found to be much less susceptible to inhibition by small AuNPs [in vivo relevance of observed in vitro inhibition for xenobiotics, in that approximately 85% of drugs in current use are primarily eliminated by these microsomal P450-catalyzed oxidations in humans [In this study we examine the influence of tannic acid-stabilized gold nanoparticles on a battery of CYP probe reactions conducted at near-d Figure . Under tll AuNPs , polystyll AuNPs , or nanoll AuNPs . Althougn humans . Interes0 value 6.04 μM, f12 particles/mL. The degree of inhibition by 15-nm AuNPs (70%) was observed to be about 3.4 times that of 7-nm AuNPs (21%). At fixed number concentration, increasing the particle radius leads to a higher mass concentration and larger NP surface area. As expected, the inhibition efficiency of 15-nm NPs is much higher than that of 7-nm NPs, owing to the larger surface area (ca. four- to fivefold) provided by the 15-nm NPs at the same particle concentration. This good agreement with the experimental results indicated that the nanogold inhibition of CYP-selective activities is highly dependent on the AuNP/protein ratio, probably via a typical noncompetitive inhibition mechanism anticipated from the protein (or NP surface area) dependence of the inhibition degree [In view of the huge variation in particle number between NPs of different sizes at the fixed mass concentration , a furthn degree . It is nn degree . Tannic n degree . As a mun degree . Howevern degree ,26 or inin vitro incubation mixture is a complex miscellaneous HLM-NP dispersion system, and AuNPs will inevitably interact with the microsomal components , cofactor (NADPH), substrates, and metabolites as well as buffering agents once involved in the incubation media. Most of all, there has been increasing evidence that NP-protein interactions play a crucial role in NP-induced conformational changes and enzymatic function impairments of the adsorbed enzymes [The above  enzymes -24. Howein vitro probe reactions substantially reduces the adsorption effects on the measurement of catalytic activity. Given the extremely low residual activity observed for CYP2D6 (3.5%) and CYP2C19 (4.3%), far below 60% and 70%, respectively, under the inhibition experiments by coincubating with 5-nm AuNPs  followed by residual CYP activity measurement by dilution into a secondary incubation containing the CYP isoform-specific substrate plus NADPH. Figure Based on the observed dose-dependent inhibition results Figure , combineIn some ways, the NP dispersion in such a complex biological medium may cause a shift in the particle size distribution or even some aggregation, which can significantly distort the available NP dose and consequently misinterpret any results obtained for NP interactions with living systems. Although the tannic acid-stabilized AuNPs are highly monodispersed with sufficient electrostatic repulsion to maintain the colloidal stability in aqueous solution  at 521 nm (in water as control) decreased dramatically  could cause a decrease of AuNP optical density and localized surface plasmon resonance. Once the NP aggregates are initialized, subsequent nanoprecipitation will occur and their monodispersion status cannot be reversed by adding proteins , but the presence of microsome or BSA in the λmax decreases significantly with time for the first 60 min of incubation, but the addition of HLM above 5 μg mL-1 protein could effectively stop the salt-induced aggregation process  and then to 186 nm (60 min) after addition of 5 μg mL-1 HLM with PBS, and it increased to 50 nm (0 min) and then to 93 nm (60 min) by incubating with 50 μg mL-1 HLM (in PBS). The NP size remained almost unchanged (at around 86 nm) during the incubation period under 0.5 μg mL-1 HLM , corresponding to the formation of a small number of aggregates. While 7-nm AuNPs exhibit the maximum red shift of λmax (17.5 nm) and maximum Dh increment (145.3 nm) when mixed with HLM at the intermediate concentration (5 μg mL-1), further confirming the possibility of strong adsorption and interactions between the microsomal membrane components and AuNP surface. In the case of higher HLM content (to 500 μg mL-1), the UV–vis spectrum is distorted in the range of 200 to 500 nm and yields a remarkably red shifted absorption band at λmax probably because too many excess interacting biomolecules decrease the surface plasmon resonance of the particles.We also used the DLS technique to monitor the number of average particle diameter and zeta-potential under real incubation conditions, which provided an orthogonal comparison to the spectroscopic methods for the direct physical characterization of molecular conjugation to AuNPs. Table M Figure . The surλmax red shift of the tannic acid-stabilized AuNPs were observed  could slow down the NP aggregation rate in buffer as mentioned above, no effects of BSA on al file 3. Despiteal file 3. The mical file 3 and in vcubation . The reccubation ,26.The mammalian CYPs are N-terminally anchored to the endoplasmic reticulum membrane with their large cytoplasmic catalytic domains in the shape of a triangular prism . Since t3, respectively [First, CYP isozymes can adsorb on AuNPs to alter protein conformations and thus cause their functional impacts, with both stabilization (activation) and inhibition (deactivation) being possible. Soft  interactions between negatively charged NPs and proteins were reported to modify the tertiary structure of proteins and enzymatic activity in a size-dependent manner . It is nectively . Interesectively -25, as win vitro microsomal incubations. It is revealed that AuNPs demonstrate differential inhibitory effects on CYP isozymes in a dose-, size-, time-, and CYP-dependent manner. The strong inhibition of 2C9, 2C19, 2D6, and 3A4 can be anticipated to be a potential source of AuNP-drug interactions for potential nanotoxicological risk assessment when co-exposed with some medicines, as these sensitive CYPs relate to clearance of the largest proportion of drugs and other xenobiotics. We also monitor the time evolution of the dispersion and surface status of AuNPs and characterize their stability and molecular conjugations during incubations. HLM or BSA can perform as a protection layer to prevent against the PBS-induced colloidal nanoaggregation in the AuNP/HLM/PBS mixtures. As expected, the observed red shift of the surface plasmon resonance peak, the drop of surface charge, and the increase in the hydrodynamic diameter further indicate the strong adsorption/interaction between the microsomal components and AuNP surface by incubating a HLM/AuNP complex. Since the nanosized particles may interfere with the CYP functions from the microsomal membrane, the NP-bilayer interactions in vesicular HLMs are important determinants of the overall enzymatic kinetics. Our findings set a basis for investigating NP-related enzyme inhibitions of membrane-bound proteins from the NP-membrane interface. Overall, by combining the activity data of CYP isozymes with direct characterization of AuNPs during the real incubations, we can further explore the potential underpinning mechanisms for controlling the enzyme activities after exposure of TA-coated AuNPs differing in size to the incubation media. Our findings add to recent data suggesting that CYP adsorption to NPs, reversible TA-CYP binding, NP adhesion to HLM membrane, and blocking cavity on the CYP surface may be supported as the major mechanisms for the inhibitory effects caused by AuNPs. Given the inherent heterogeneity and dynamic nature of biomembranes, however, it is difficult to distinguish the propensities for AuNPs to attach onto the CYP protein molecules or disrupt the vesicle HLM membranes. For membrane-bound enzymes, interference from complex nanoparticle-membrane interactions (due to membrane curvature or vesicle rupture) can skew results and complicate the interpretation of the nanoparticle-induced effects on protein functions unless additional steps are taken to remove it. Further studies with complementary experimental approaches and multidimensional in situ measurements of the microsome-nanoparticle hybrids will explore a more complete picture of the interaction patterns and molecular dynamics toward a deeper comprehension of the complex NP-induced inhibitory effects. Although physical disruption of microsomal membranes from AuNPs interacting at surface sites cannot be totally eliminated as a possibility, the differential sensitivity of the CYP isozymes to the inhibition suggests that steric blocking effects on the substrate pockets of the isozymes may also offer a plausible explanation to the observed large discrepancy in the inhibitory efficiency. At this point, more work is required to determine the possible and dominant mechanisms of microsome-nanoparticle interaction processes and the extent to which they contribute to in vivo nanotoxicology, which will bear importantly upon our understanding of the potential risk and hazard related to nanomaterials.Many types of nanomaterials are reported to have inhibitory effects on different kinds of enzymes including cytochromes P450s. However, comprehensive investigation of the influence caused by metallic nanoparticles on CYP structures and their maker reactions is mostly an unexplored area. Since there is no data available on the relevance of the concentrations used for human exposure, in this mechanistic study, we conduct a systematic investigation of the impacts of AuNPs with different particle sizes and concentrations on five major human CYPs under normal 4: magnesium sulfate; MRM: multiple reaction-monitoring; NPs: nanoparticles; NaCl: sodium chloride; NADPH: β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate; NaH2PO4: sodium dihydrogen phosphate; Na2HPO4: disodium hydrogen phosphate; PBS: phosphate buffer; Q1: precursor ion; Q3: product ion; QC: quality control; RSD: relative standard deviation; SPR: surface plasmon resonance; TA: tannic acid; TEM: transmission electron microscope; UV: ultraviolet.AuNPs: gold nanoparticles; BSA: bovine serum albumin; CE: collision energy; CXP: collision cell exit potentials; CYP: cytochrome P450; DLS: dynamic light scattering; DP: declustering potential; ESI: electrospray ionization; HLM: human liver microsome; IS: internal standard; LC-MS: liquid chromatography-mass spectrometry; MgSOThe authors declare that they have no competing interests.MY carried out the LC-MS assays, CYP inhibition studies, bio-sample preparation and drafted the manuscript. LT performed the characterizations of UV–vis spectroscopy, digital photography, TEM, and DLS measurements. JZ carried out the incubation experiments, results discussion, and analysis data. ML performed nonspecific binding experiments and participated in writing the manuscript. BG conceived of the study, designed, and coordinated the project and drafted the manuscript. YL participated in DLS and TEM measurements. BC participated in design of the study and helped to review the manuscript. All the authors read and approved the final manuscript.Figure S2. BSA-induced alteration in UV–vis extinction spectra of AuNPs and absorbance value at λmax when initial incubating (at 0 min) with different BSA concentrations. Table S1. Technical data sheet of PELCO tannic BioPure™ gold nanoparticles. Technical data sheet of physicochemical characteristics of PELCO tannic BioPure™ gold nanoparticles investigated. Table S2. Experimental conditions for microsomal CYP assays. Preparation of stock and working solution of substrates, incubation conditions, and analytical parameters for assays.The optical properties of tannic acid-coated AuNPs. Size-dependent optical properties of tannic acid-coated AuNPs at mass concentration of 1 mg/mL and 25 μg/mL (top); a comparison of the UV/Vis spectra of gold colloid in 6 months of storage (bottom). The UV–vis absorption spectra with the centered bands were consistent with well-dispersed, spherical AuNPs in aqueous media. Click here for file


In order to access MPBs, polar structures with distinct polarization directions away from [111]p in these Bi-based families are required.Piezoelectrics are key functional materials in actuator and sensor applications.3 (LFO)–La2MgTiO6 (LMT) where the introduction of LFO (tolerance factor t=0.95) would alter the average structural asymmetry on the A site by combining the aspherical Bi3+ and spherical La3+ cations, and the addition of Mg2+ and Ti4+ from LMT (t=0.95) on the B site would reduce the dielectric loss caused by the increase in Fe3+ content. LFO adopts the Pmnb structure of GdFeO3 with an a+b−b− tilt system and 0.18 Å antiferrodistortive La3+ displacements along the [110]p direction. LMT (P21/a) has the same A-site displacement pattern and tilt system plus rock salt ordered B-site cations.7We investigated the pseudoternary phase field BFTM–LaFeOt=0.96) is the only material with the rhombohedral R3c space group, and is surrounded by a two-phase region. At low LFO content and along the BFTM–LMT line, an orthorhombic perovskite phase and an Aurivillius phase coexist BFTM–xLFO line in Figure Pmnb BLFTM samples are non-centric , whereas the Pmnb materials  are centric as expected. Thin films can be grown by pulsed laser deposition .Phases in the BFTM–LMT–LFO (BLFTM) field Figure  were pre0.72La0.28(Fe0.46Ti0.27Mg0.27)O3 (0.72BFTM–0.28LFO) composition. Of the candidate space groups which gave similar model-independent Lebail fits in the observed 2pa× pa× pa cell only Pmnb and Pmc21 give acceptable Rietveld refinements of neutron  , with the non-polar Pmnb model unable to satisfactorily fit the (110), (200), and (011) reflections  Figure  and syncs Figure .Pmc21 structure of 0.72BFTM–0.28LFO  is adopted at low temperature by the high-pressure form of the B-site-driven CdTiO3 ferroelectric,33.a  direction  antiferrodistortive displacements at both A sites along b and c and at the B site along a  Pmnb phase can be understood in terms of distortion modes derived from the pm a aristotype cell, evaluated with the IsodisplacePmnb ,}, origin=) is produced by the R4+ ,}, origin=) is driven by the onset of the dominant Γ4− polar displacement at the A and B sites  phase of BaTiO33.5+ mode) present in the Pmnb precursor affords the 0.31 Å displacement at the A1 site, while the out-of-phase combination gives the 0.14 Å polar displacement at the A2 site along the [110]p direction. The small A-site antiferrodisplacive R5+ mode .δ, suggesting that LMT is particularly effective in reducing the loss. All the Pmc21 [110]p displaced materials have higher permittivities than [111]p displaced rhombohedral BFTM, and the broad step-like decrease in permittivity for BFTM above 1 kHz, suggestive of a relaxation phenomenon, is not observed in the new polar phase. The magnitude of the relative permittivity rules out the possibility of contributions from extrinsic charge carriers at grain boundaries or electrode–sample interfaces.21Electrical properties of BLFTM were measured on 97+% dense ceramics, with dielectric loss reduced by the addition of 0.2 wt % MnOPmc21 0.72BFTM–0.28LFO   hysteresis loop before dielectric breakdown, which is a common problem in BiFeO3 ceramics.P(E) loop came from the Pmc21 structure composition 0.625BFTM–0.25LFO–0.125LMT  for 0.72BFTM–0.28LFO .p displacements in the present BLFTM case may arise from the presence of Mg2+ and particularly Ti4+ on the B site in the BFTM parent, as Ti4+ produces ferroelectric [110]p displacements in O BaTiO3.p antiferrodisplacive motions and the dominant ferroelectric displacements along the same direction driven by Bi3+ and Ti4+, reflecting structural frustration between the bonding preferences of the two A-site cations. In the present BLFTM system, the O′ structure is separated by two-phase regions involving non-perovskite impurities from the R′ structure, and bridging this phase gap is important in the search for enhanced functionality associated with potential phase boundaries differing in polarization direction.Figure


Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of  Hedgehog proteins are critical regulators of embryonic tissue growth and organization in species ranging from flies to humans. Binding of the secreted Hh protein to its receptor at the surface of cells triggers an intracellular signal that is initiated by Smoothened (Smo). Upon exposure of cells to Hh, Smo becomes active and signals through a series of downstream proteins to regulate gene expression. Although Smo proteins in flies and mammals are similar, the critical regions involved in activation and signal initiation differ between the two, implying that different mechanisms have evolved in different organisms. Using the fruit fly as a model organism, we identified regions in Smo that are phosphorylated by a protein kinase called Gprk2 to enhance Smo activity. These phosphorylation sites overlap with previously identified sites in mouse Smo and are conserved in Smo proteins in many animals. Phosphorylation at these sites regulates the recruitment of Costal2 to Smo, a critical step in signal initiation, through a region of the protein that is also highly conserved. Our results indicate that Gprk2 phosphorylation represents an evolutionarily ancient and conserved mechanism for regulating Smo activity, and suggest that Smo regulation and signaling are more similar between different species than previously thought. Drosophila) or primary cilium , and activates downstream signaling, leading to stabilization of Ci/Gli family transcription factors and Hh target gene expression The Smoothened (Smo) family of seven-pass transmembrane proteins initiate cytoplasmic Hedgehog (Hh) signaling. Smo proteins are activated by multisite phosphorylation in the cytoplasmic C-terminal tail, which counteracts the electrostatic effects of adjacent clusters of positively charged residues thought to maintain the protein in an inactive conformation. Upon phosphorylation, Smo undergoes a conformational change, dimerizes, and accumulates at the plasma membrane  and Casein kinase I (CKI) sites located in the Smo autoinhibitory domain (SAID) is necessary and sufficient for activation Drosophila Smo are conserved in vertebrate Smo proteins; instead G-protein-coupled receptor kinase (GRK) 2 and CKI are the principal kinases that activate vertebrate Smo proteins by phosphorylating them at a different set of sites Although the extracellular N-terminus and seven-transmembrane regions of Smo are highly conserved, only the first 100 amino acids of the cytoplasmic C-terminus is broadly conserved. The more distal C-terminus, in many cases hundreds of amino acids long, is completely divergent in different phyla. Because they target different portions of the C-terminus, the phosphorylation mechanisms regulating Smo differ fundamentally between invertebrates and vertebrates. In Drosophila and vertebrates, Smo binds to the downstream effector Cos2 and its orthologue Kif7, respectively Drosophila Smo C-terminus Drosophila and vertebrate Smo signal through similar effectors The means by which Smo engages the downstream signaling machinery through its C-terminus also appears to have diverged. In Drosophila. Gprk2, one of the two Drosophila GRKs, is required for Smo to drive high-threshold Hh target gene expression gprk2 causes a reduction in cyclic AMP (cAMP) levels, affecting PKA-dependent Smo activation. Target gene expression can be largely rescued in gprk2 mutants by increasing cAMP levels, suggesting that this indirect effect of Gprk2 on Smo plays an important role in Hh pathway function GRKs are also implicated in Smo activation in To further explore the effects of direct Gprk2 phosphorylation on Smo activity, we mapped four new clusters of Gprk2-dependent phosphorylation sites in the cytoplasmic C-terminus. We find that mutation of these sites to non-phosphorylatable residues reduces Smo dimerization and its ability to promote Hh target gene expression. Phosphomimetic mutations bypass the requirement for Gprk2. Importantly, the phosphosites we mapped overlap with CKI/GRK sites mapped in the mouse Smo C-terminus, and are remarkably well-conserved in Smo orthologues throughout the bilaterian lineages. We demonstrate that the evolutionarily conserved core of Smo is a functional, GRK-regulated protein that is sufficient to activate downstream signaling, suggesting that all bilaterian Smo proteins share a common regulatory and signaling mechanism.SDgprk2 depletion on cAMP levels, there was a substantial reduction of Smo phosphorylation (detected by increased mobility in SDS-PAGE) upon dsRNA-mediated depletion of Gprk2 that could not be accounted for by the GPS sites (SD.GPSA12) had only a small effect on Smo signaling activity SD and SmoSD.GPSA12 had similar ability to drive expression of the highest-threshold Hh target gene, anterior engrailed (en), in vivo (We previously reported that Gprk2 affects phosphorylation of Smo at sites other than GPS1 and GPS2 PS sites . Previou in vivo . These rcore-GFP), which retains the highly conserved core of the protein but lacks more than 75% of the cytoplasmic tail including all three clustered PKA/CKI sites, still shifted in response to gprk2 depletion (Δ603-GFP) eliminated this response (core to non-phosphorylatable Ala residues (Smocore.c1-3A-GFP) eliminated Gprk2-dependent phosphorylation , and at three of five sites  in cluster 3  are phosphorylated in response to Hh To look at Smo phosphorylation more directly, we used a label-free quantitative liquid chromatography/tandem mass spectrometry (LC-MS/MS) approach to compare the relative levels of phosphorylated:non-phosphorylated forms of individual Smo phosphopeptides in control and ells see  [21]. Coluster 3 . For eac675, Ser679, Ser682) located in the SAID (SD.c1-3A background resulted in a protein (SmoSD.c1-4A-GFP) that no longer shifted in response to gprk2 depletion (741 and Thr742) in gprk2-depleted cells  transcriptional reporter assays gprk2 depletion using a mix of dsRNAs targeting the gprk2 5′- and 3′-untranslated regions (UTRs) significantly reduced both Smo phosphorylation and ptc-luc reporter activity in SmoSD-GFP transfected cells  reduced ptc-luc reporter activity by 80% compared to SmoSD-GFP, but did not eliminate it (SD.c1A) or six cluster 2 (SmoSD.c2A) sites reduced SmoSD-GFP-driven ptc-luc reporter transcription, but both were significantly more active than SmoSD.c1-4A-GFP (SD.c3A) had less effect, while mutation of the three sites in cluster 4 (SmoSD.c4A) had no significant effect on activity  showed only a partial resistance to depletion of the kinase  restored Hh responsiveness (c1-4A-GFP) strongly impaired this rescue (SD. Unlike PKA and CKI phosphorylation c12D-GFP was as dependent on Hh as SmoWT-GFP (SA.c12D)  . These rSD dimerization in intact cells. In control experiments, co-expression of SmoSD molecules C-terminally-tagged with GFP10 (SmoSD-GFP10) and Renilla luciferase II (SmoSD-Luc) yielded a net BRET signal between the two which responded to Gprk2. Gprk2 overexpression, which increased SmoSD-GFP phosphorylation  throughout the developing wing disc using the nub-GAL4 driver strongly suppressed Hh signaling, leading to loss of the central region of the adult wing patterned by Hh  but not high (en) threshold target gene expression . In contrast, Smoc1-4A-GFP accumulated ectopically in Hh-responding cells (bracket), as endogenous Smo does in gprk2 mutants Interestingly, the cluster 1–4 Ala-substituted form of Smo faithfully phenocopied the effects of Gprk2 loss on Smo accumulation. In discs depleted of endogenous Smo, wild-type Smo-GFP accumulated in the normal pattern ng cells , bracketDrosophila Smo - see Drosophila Smo, including the SAID, PKA/CKI sites, and binding sites for the kinesin-like protein Cos2 Broad sequence conservation in the cytoplasmic C-terminus of Smo is limited to the membrane-proximal 100 amino acids , respectively   , as wellld-type) . It also and ptc , produci  or strongly inhibited by Hh e.g. . In cont highest . This pactivated , indicatDrosophila and vertebrates, Smo binds to Cos2 and its orthologue Kif7, respectively core recruits Cos2, we adapted previously-described FRET biosensors core interaction by BRET. Co-expression of Smocore C-terminally-tagged with GFP10 (Smocore-GFP10) and Cos2 C-terminally tagged with Renilla luciferase II (Cos2-Luc) yielded a net BRET signal that was responsive to Gprk2. Gprk2 overexpression increased Smocore-Cos2 BRET more than 2.2-fold, whereas gprk2 depletion reduced it by 60%   . These rcore. To see if the Cos2 recruitment activity we observed was due to binding between amino acid 651 and the end of Smocore at amino acid 663, we made a further truncation to amino acid 651 , just N-proximal to Gprk2 phosphorylation cluster 2  are phosphorylated by Gprk2, with Ser604, Thr610, and Thr612 being further validated using phosphospecific antisera. Two of the sites in cluster 3 (Thr551 and Thr555) are likely not Gprk2 targets, as we did not detect phosphorylation at either site in control cells. We did not obtain peptide coverage in the region containing the remaining six sites , which make up cluster 2; however, phosphorylation at four of these sites has been observed by others gprk2-depleted cells. In mSmo, CK1 phosphorylates the sites corresponding to cluster 1 whereas GRK2 phosphorylates cluster 2 Drosophila Smo  are capable of activating most target gene expression in vivo. Activity above this level may simply be non-physiological.Gprk2 regulates Smo stability and activity, with both effects mediated at least partly through direct phosphorylation. Several observations indicate that Gprk2 directly enhances Smo activity in Hh-responding cells. The extent of the conformational shift that Smo undergoes upon activation is lower in en, which was only partially activated, independent of Gprk2. Thus full PKA/CKI phosphorylation is sufficient to strongly, but not completely, activate Smo. Full activation requires phosphorylation by Gprk2.Although Gprk2 phosphorylation contributes to activating Smo, it is neither necessary nor sufficient. Smo activation appears to be a two-step process, with phosphorylation by PKA and CKI in the SAID serving as the principal trigger . SAID phMimicking Gprk2 phosphorylation alone had no effect on Smo activity in the absence of Hh, nor could it activate in the absence of phosphorylation by PKA/CKI. This could be because access to the Gprk2 sites is blocked without prior PKA/CKI phosphorylation. However, it seems more likely that the effect of PKA/CKI in controlling accumulation of Smo at the cell surface, where GRKs are typically localized core protein that lacks both the SAID domain and distal C-terminus. These observations favour a model in which Gprk2 phosphorylation more directly affects the conformation of the proximal C-terminus or seven transmembrane domain portion of Smo to enhance its activity.PKA and CKI phosphorylation disrupts electrostatic interactions between the SAID domain and the distal C-terminus, thereby promoting a more open and active Smo conformation c1-4A mutant accumulated ectopically in wild-type Hh-responding cells, as endogenous Smo does in gprk2 mutants, demonstrating that Gprk2 triggers Smo turnover by directly phosphorylating it. This is consistent with the typical role of GRKs in receptor desensitization, and supports the conclusion that Gprk2 phosphorylation limits the duration of Smo signaling Previous studies have shown that Gprk2 promotes Smo internalization and degradation in response to Hh Drosophila Smo and GRK2/CKI phosphorylation of mSmo Drosophila Smo.The striking conservation of the first two Gprk2 phosphorylation clusters in all bilaterian Smo proteins clearly points to an ancient origin and common function. Indeed, there are some parallels between the functions of Gprk2 phosphorylation of core protein  express target genes at lower levels than Hh-responding cells at the A/P boundary. Ptc overexpression in S2 cells reduces Smocore-driven ptc-luc reporter expression (not shown). Because Ptc downregulates full-length Smo through a mechanism involving ubiquitination of the SAID core could explain its accumulation in far A cells. How Ptc regulates Smocore activity is unclear, but could be related to its proposed function in regulating the levels of a Smo agonist or antagonist One important difference is that GRK phosphorylation is required and sufficient for Smo activation in mammals but not flies. In this regard, mSmo behaves more like the truncated Smo protein . Our anaDrosophila and mammalian Smo proteins involves Cos2/Kif7. Despite lacking previously mapped Cos2 interaction domains, Smocore is capable of recruiting Cos2, and the ability of successive C-terminally truncated forms of Smocore to do so correlates with their ability to stimulate target gene expression. This truncation approach allowed us to identify a region required for Smo-Cos2 interaction between amino acids 625–651. Gprk2 phosphorylation cluster 2 falls within this region, and the ability of Smocore to recruit Cos2 is strongly influenced by Gprk2 phosphorylation. Phosphorylation may influence Smocore conformation in a way that favours Cos2 interaction. Alternatively, Cos2 may interact with this region preferentially in a phosphorylated state, as β-Arrestins do with GRK-phosphorylated GPCRs Signaling downstream of both core provides some insights into the potential evolutionary origin of Smo. Smocore is a highly conserved, minimal functional form of Smo, and we speculate that it may closely resemble the ancient form of Smo in the common bilaterian ancestor. It displays a mode of regulation that is typical of GPCR desensitization, suggesting that the ancient form of Smo may have behaved more like a classical GPCR. The evidence suggests that different mechanisms have evolved in different lineages for restricting the activity of Smocore, in the form of C-terminal negative regulatory domains. In vertebrates, a stretch of positively charged residues in the C-terminus serves to keep the core in an inactive conformation, and phosphorylation primarily at the first two GRK/CKI clusters overcomes this effect. This same phosphorylation mechanism has been retained in Drosophila and other arthropods. However, through evolutionary time, this group appears to have acquired a PKA/CKI-regulated autoinhibitory domain that has come to dominate Smo activity. In contrast to vertebrates, the ancestral GRK mechanism has been relegated to a modulatory role in flies, where it is required to achieve the highest level of Smo signaling. The principal role of driving Smo into an open conformation and activating downstream signaling has been assumed by PKA/CKI phosphorylation of the SAID domain. One consequence of SAID phosphorylation is recruitment of Cos2 and Fu to binding sites in the nonconserved distal C-terminus, leading to Cos2-dependent Fu dimerization and activation core. Further analysis of Smocore will be necessary for a full understanding of how Drosophila Smo connects to the downstream signaling apparatus, and should provide insights into a signaling mechanism common to all Smo proteins.Our analysis of SmoSDEcoRV site. A 1023 nt EcoRV-EcoRI fragment of wild-type or SmoSD coding sequence containing codon 458–798 and harboring all Gprk2 and PKA phosphorylation sites was subcloned into pBluescript (pBS). The resulting constructs were used as templates for multiple rounds of PCR-based site-directed mutagenesis in order to mutate all Gprk2 sites. The modified EcoRV-EcoRI fragments were then cloned back into full-length Smo expression plasmids. To generate Smo truncations  Smo sequences between the EcoRV site at codon 458–459 and the indicated 3′ codon were PCR amplified, introducing a 3′ NotI site. The resulting EcoRV-NotI fragments were cloned into Smo expression vectors. All Smo constructs were C-terminally tagged with either GFP, GFP10 or RLucII. The tags were engineered as a cassette flanked by NotI and KpnI restriction sites. Coding fragments were cloned into expression constructs for use in cell culture  and flies (pUAST-AttBUAS-smo-3′UTR-dsRNA was generated by cloning a genomic PCR-generated fragment containing nucleotides 2L:281756..281981 of the smo 3′-UTR. The 226 nt long fragment was cloned between the EcoRI-AvrII sites and in the opposite orientation between the NheI-XbaI sites of pWIZ (Drosophila Genomics Resource Centre). To generate catalytically inactive forms of Gprk2, point mutations changing Lys338/339→Met (Gprk2kd1453→Asn (Gprk2kd2) were generated by PCR mutagenesis and cloned downstream of a Myc-epitope tag in pRmHa3.puro. A C-terminal luciferase-tagged Cos2 expression construct used in BRET assays was engineered by flanking the Cos2 coding sequence at the 5′ and 3′ end with an EcoRI and a NotI site, respectively. The resulting EcoRI-NotI fragment was cloned into pRmHa3.puro. The RLucII cassette described above was cloned downstream of the Cos2 sequence at the NotI site. Fu coding sequence was cloned downstream of a Myc-epitope tag in pRmHa3.puro. Sequences of all constructs were verified. For experiments involving Hh treatment, cells were co-transfected with NpRmHa3.puro/HhDrosophila Hh. For ptc-luc reporter assays a mixture of the following constructs was used: pRmHa3/Ci , pGL.basic/ptcΔ136-lucpRL/CMV (Promega).For expression of Smo mutants, we first silently mutated codons 458 and 459 of wild-type and SmoGprk2kd1 or Asp45UAS-Smo variant transgenic fly strains were generated by recombining the appropriate pUAST-attB transgenes into the 65B2 attP locus using the PhiC31 system 3′UTR-dsRNAUAS-smo transgene were generated by standard P-element-mediated transgenesis. Other fly strains and their sources: del1gprk2 and KOgprk2ap-GAL4, nub-GAL4, 10638dpp (dpp-LacZ), UAS-Dicer, tstubP::GAL80 were from the Bloomington Drosophila Stock Center.All 604 and anti-pThr610/pThr612 phosphospecific antisera were generated by GenScript. Rabbits were immunized with phosphorylated peptides (KGRL{pS}ITLYNTHC or CSITLYN{pT}H{pT}DPVGL), and antibody was isolated from serum by modified peptide affinity column purification and unmodified peptide cross-adsorption. Anti-Smo antibody was raised in guinea pigs against the same His-tagged fragment of the Smo C-terminus (amino acids 560–1036) as previously used Anti-pSerSD variants, flies were mated at 25°C and 0–48 h old offspring transferred to 29°C to inhibit GAL80ts and activate apGAL4-dependent transgene expression. For experiments involving rescue of dsRNA-mediated Smo depletion, crosses included a UAS-Dcr transgene and were carried out at 27°C to maximize the smo dsRNA phenotype while minimizing the ectopic effects of transgenic Smo overexpression. For experiments in a KO/gprk2del1gprk2 mutant background, flies were mated at 25°C and 0–48 h old offspring transferred to the restrictive temperature of 29°C For expression of SmoUAS-Dcr/+;nub-GAL4/+3′UTR-dsRNAUAS-Dcr/UAS-GFP;nub-GAL4/UAS-smo3′UTR-dsRNA;UAS-SmoWT/+UAS-Dcr/+;nub-GAL4/UAS-smo3′UTR-dsRNA;UAS-Smoc1-4A/+UAS-Dcr/+;nub-GAL4/UAS-smo3′UTR-dsRNA;UAS-Smocore/+UAS-Dcr/+;nub-GAL4/UAS-smo10638/UAS-smo3′UTR-dsRNAUAS-Dcr/UAS-GFP;ap-GAL4,dpp10638/UAS-smo3′UTR-dsRNA;UAS-SmoWT-GFP/+UAS-Dcr/+;ap-GAL4,dpp10638/UAS-smo3′UTR-dsRNA;UAS-Smoc1-4A-GFP/+UAS-Dcr/+;ap-GAL4,dppSD-GFP/tubP::GAL80tsap-GAL4/+;UAS-SmoSD.c1-4A-GFP/tubP::GAL80tsap-GAL4/+;UAS-SmoSD-GFP,gprk2del1/gprk2KOap-GAL4/+;UAS-SmoSD.c12D-GFP,gprk2del1/gprk2KOap-GAL4/+;UAS-Smo10638/UAS-smo3′UTR-dsRNA;UAS-Smocore-GFP/+UAS-Dcr/+;ap-GAL4,dpp10638/UAS-smo3′UTR-dsRNA;UAS-Smocore.c1-3A-GFP/+UAS-Dcr/+;ap-GAL4,dppcore-GFP,gprk2del1/gprk2KOap-GAL4/+;UAS-SmoDrosophila Schneider's medium (Lonza) supplemented with 10% fetal bovine serum (Gibco) and 50 U/ml penicillin and streptomycin (Gibco). Exceptionally, experiments for Drosophila S2 cells adapted to growth in serum-free medium , which show more pronounced Smo phosphoshifts due to a higher basal level of phosphorylation in vitro transcription using templates PCR-amplified from genomic DNA . β-Gal dsRNA was used as a control. Forward and reverse primers included T7 (5′-TAATACGACTCACTATAGGGAGA-3′) and T3 (5′-AATTAACCCTCACTAAAGGGAGA-3′) promoter sequences, respectively. Top and bottom strand RNAs were generated using MEGAscript T7 and T3 in vitro transcription kits, mixed in equal amounts, and heated to 95°C followed by slow cooling to room temperature to anneal.Most experiments were performed using S2-R+ cells grown in 6 cells were typically plated on day 1 in 24 well plates in 0.5 ml of complete Schneider's medium and each well was transfected with 100–250 ng of the indicated pRmHa.puro expression constructs using X-tremeGENE HP transfection reagent (Roche) according to manufacturer's instructions. On day 2, the cells of each well were split into 2 new wells of a 24 well plate and treated with 5 µg of the indicated dsRNA. On day 3 to 4, a second dose of dsRNA was applied and transgene expression was induced by addition of CuSO4 to a final concentration of 0.5 mM. Cells were harvested and processed on day 7.For biochemical analysis, <1×10ptc-luc reporter assays, S2-R+ cells were transfected in 24 well plates on day 1 of the experiment as described above. 100 ng pRmHa/Ci, 75 ng pGL.basic/ptcΔ136-lucpRL/CMV, and 100 ng of each additional expression plasmid  were typically used. Total DNA amounts in the transfection mix were normalized using empty pRmHa.puro vector. On day 2 the cells were split into 4 wells of a 96 well plate and each well was treated with 0.5–1 µg dsRNA. On day 3 or 4 transgene expression was induced by addition of CuSO4 and a second dose of dsRNA was administered. Cells were processed on day 7 and luciferase activity measured using the Dual Luciferase Reporter system (Promega) according to manufacturer's instructions. Assays were performed at least two times in quadruplicate, and the data was pooled. Statistical significance was assessed using two-tailed Student's t-tests.For SD-RLucII variant, 300 ng of the Smo-GFP10 variant and 100 ng of mycGprk2 (if applicable) per well of a 24-well plate. For BRET assays monitoring recruitment of Cos2 75 ng of Cos2-RLucII, 300 ng of the indicated Smo-GFP10 variant, and 75 ng mycFu plasmids were transfected. Cells were re-plated in 4 wells of a white-walled 96-well plate and subjected to dsRNA treatments and transgene induction as described above. BRET measurements were performed on day 7 as previously described For bioluminescence resonance energy transfer (BRET) experiments measuring Smo dimerization S2-R+ cells were transfected with 100 ng of the Smog and 4°C. Anti-GFP mAb agarose  was added to soluble extracts and samples incubated on ice for 2 h. Beads were washed 2–3 times in 1 volume of lysis buffer and precipitated proteins extracted by addition of 1× SDS-PAGE sample buffer and heating at 75°C for 6 min. For most experiments, proteins were fractionated by SDS-PAGE on standard polyacrylamide gels. For S2 cells expressing Smo-GFP variants were lysed in lysis buffer  for 15 min on ice. Insoluble material was removed by microcentrifugation for 15 min at 12,000× SD-GFPpRmHa3.puro/Smo as above. A day later, medium was replaced and 20 µg/well control  or gprk2 dsRNA was added to the cells. After three days of growth, the cells were harvested and replated in a 10-cm plate, along with 100 µg/plate of the appropriate dsRNA. SmoSD expression was induced by addition of 0.5 mM CuSO4. 2–3 d later, cells were washed with ice-cold PBS and lysed in 3 ml RIPA buffer for 15 min on ice. Lysates were cleared by microcentrifugation for 15 min at 12,000× g and 4°C. SmoSD-GFP was immunoprecipitated using anti-GFP mAb agarose for 2 h at 4°C with rotation. Beads were washed 4 times with ice-cold RIPA buffer before addition of 1× SDS-PAGE sample buffer and heating at 75°C for 6 min. Samples were frozen at −80°C and typically 2 to 3 such preps were pooled for subsequent analysis. Pooled samples were fractionated by SDS-PAGE on 4–15% polyacrylamide gradient gels (BioRad), stained using Colloidal Blue (Life Technologies) according to manufacturer's protocol, and the band corresponding to Smo was excised from the gel.S2-R+ cells were plated in 1 to 3 wells per condition of a 6-well plate and transfected with 2.5 µg/well of Gel pieces were washed with water for 5 min and destained twice with the destaining buffer  for 15 min. An extra wash of 5 min was performed after destaining with a buffer of ammonium bicarbonate (50 mM). Gel pieces were then dehydrated with acetonitrile. Proteins were reduced by adding the reduction buffer  for 30 min at 40°C, and then alkylated by adding the alkylation buffer  for 20 min at 40°C. Gel pieces were dehydrated and washed at 40°C by adding ACN for 5 min before discarding all the reagents. Gel pieces were dried for 5 min at 40°C and then re-hydrated at 4°C for 40 min with enzyme solution. Tryptic digestion was performed with a 6 ng/µl solution of sequencing grade trypsin from Promega in 25 mM ammonium bicarbonate buffer, incubated at 58°C for 1 h and stopped with 15 µl of 1% formic acid/2% acetonitrile. Chymotryptic digestion was performed with a 40 ng/µl solution (Roche) in 100 mM Tris HCl- 25, mM CaCl2, pH 8 buffer, incubated at 25°C for 4 h and stopped with 15 µl of 1% formic acid/2% acetonitrile. Supernatant was transferred into a 96-well plate and peptide extraction was performed with two 30-min extraction steps at room temperature using the extraction buffer (1% formic acid/50% ACN). All peptide extracts were pooled into the 96-well plate and then completely dried in vacuum centrifuge. The plate was sealed and stored at −20°C until LC-MS/MS analysis. Protein digestion with Asp-N was performed in solution on tryptic digests. Samples were re-solubilized in a 50 mM ammonium bicarbonate buffer and 1 ng of Asp-N was added to each sample. Samples were incubated at 37°C for 3 h.Prior to LC-MS/MS, peptide extracts were re-solubilized under agitation for 15 min in 11 µl of 0.2% formic acid and then centrifuged at 2000 rpm for 1 min. The LC column was a C18 reversed-phase column packed with a high-pressure packing cell. A 75 µm i.d. Self-Pack PicoFrit fused silica capillary  of 15 cm length was packed with the C18 Jupiter 5 µm 300 Å reverse-phase material . This column was installed on the Easy-nLC II system  and coupled to the LTQ Orbitrap Velos  equipped with a Proxeon nanoelectrospray ion source. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). During the first 12 min, 5 µl of sample were loaded on column with a flow of 600 nl/min and, subsequently, the gradient went from 2–80% buffer B in 60 min at a flow rate of 250 nL/min and then came back at 600 nL/min to 2% buffer B for 10min. LC-MS/MS data acquisition was accomplished using an eleven scan event cycle comprised of a full scan MS for scan event 1 acquired in the Orbitrap which enables high resolution/high mass accuracy analysis. The mass resolution for MS was set to 60,000 (at m/z 400) and used to trigger the ten additional MS/MS events acquired in parallel in the linear ion trap for the ten most intense ions. Mass over charge ratio range was from 360 to 2000 for MS scanning with a target value of 1,000,000 charges and from ∼1/3 of parent m/z ratio to 2000 for MS/MS scanning with a target value of 10,000 charges. The data–dependent scan events used a maximum ion fill time of 100 ms and 1 microscan. Target ions already selected for MS/MS were dynamically excluded for 25 s. Nanospray and S-lens voltages were set to 0.9–1.8 kV and 50 V, respectively. Capillary temperature was set to 225°C. MS/MS conditions were: normalized collision energy, 35 V; activation q, 0.25; activation time, 10 ms.SD sequence using Mascot 2.3 (Matrix Science). The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.6 Da, respectively. Search parameters allowed for up to two missed enzyme cleavages. Oxidation of methionine and phosphorylation of serine, threonine and tyrosine were allowed as variable modifications while carbamidomethyl was set as a fixed modification. Matches for phosphopeptides were validated manually. In a few cases , the phosphopeptides were not confirmed by MS2, but were detected in full scan with mass accuracies of less than 2 ppm, and eluted with very similar retention times to other phosphospecies of the same peptide. Peptides were quantitated by manual integration of precursor ion LC spectra using Qual Browser  The peak list files were generated with extract_msn.exe  using the following parameters: minimum mass set to 600 Da, maximum mass set to 6000 Da, no grouping of MS/MS spectra, precursor charge set to auto, and minimum number of fragment ions set to 10. MS/MS spectra were queried against the SmoHomo sapiens (NP_005622.1), Mus musculus (NP_795970.3), Danio rerio (NP_571102.1), Paracentrotus lividus (AEX61000.1), Platynereis dumerilii (ADK38671.1), Drosophila melanogaster (NP_523443.1), Apis mellifera (XP_395373.3), Tribolium castaneum (NP_001127850.1), Daphnia pulex (EFX80809.1).Multiple sequence alignment of full-length Smo proteins from nine bilaterian animal species was generated with Clustal-Omega. The species and accession numbers corresponding to the sequences used were: Figure S1SD-GFP or SmoSD.GPSA12-GFP, with or without Gprk2 depletion. The blot was probed with anti-GFP antibody to visualize tagged Smo protein. All bands are from the same exposure of a single blot with intervening lanes removed. Although SmoSD.GPSA12-GFP has putative Gprk2 phosphorylation sites mutated to nonphosphorylatable Ala, it still undergoes a similar phosphoshift as SmoSD-GFP in response to depletion of the kinase. (B and C) Confocal micrographs of wing discs with Smo variants - SmoSD-GFP (B) or SmoSD.GPSA12-GFP (C) - expressed in the dorsal compartment using ap-GAL4. Discs were immunostained to reveal En expression. Yellow dotted lines: A/P compartment boundaries based on domains of Ci expression (not shown). Both SmoSD-GFP and SmoSD.GPSA12-GFP drive comparable ectopic expression of En in dorsal anterior cells (arrowheads) - compare to wild-type ventral anterior cells. Genotypes: SD-GFP/tubP::GAL80tsap-GAL4/+;UAS-Smo (B); SD.GPSA12-GFP/tubP::GAL80tsap-GAL4/+;UAS-Smo (C).GPS1 and GPS2 are not the principal Gprk2 phosphorylation sites in Smo. (A) Western blot analysis of GFP immunoprecipitates from S2 cells expressing Smo(TIF)Click here for additional data file.Figure S2SD-GFP or SmoSD.c1-3A-GFP, with or without gprk2 depletion. The blot was probed with anti-GFP antibody to visualize tagged Smo protein. All bands are from the same exposure of a single blot with intervening lanes removed. SmoSD.c1-3A-GFP migrates as a tighter band than SmoSD-GFP in control cells, suggesting that it is less phosphorylated. However, it still undergoes a phosphoshift in response to depletion of the kinase, indicating that additional Gprk2 phosphorylation sites exist.Additional Gprk2 phosphorylation sites outside of clusters 1, 2, and 3 exist in Smo. Western blot analysis of GFP immunoprecipitates from S2 cells expressing Smo(TIF)Click here for additional data file.Figure S3SD-GFP-driven ptc-luc reporter activity in gprk2-depleted S2-R+ cells. Treatment of cells with gprk2 5′- and 3′-UTR dsRNAs reduced ptc-luc reporter activity, and this was fully rescued by re-expressing wild-type Gprk2 but not kinase-dead Lys338/339→Met (kd1) or Asp453→Asn (kd2) mutants of Gprk2. (B) Western blot analysis of GFP immunoprecipitates (top) or total-cell lysates (bottom) of S2 cells with or without gprk2 depletion, transfected with SmoSD-GFP along with empty vector (−) or various forms of Myc-tagged Gprk2. The blots were probed with anti-GFP (top) or anti-Myc (bottom) antibodies. Re-expression of wild-type (lane 4) but not kd1 (lane 6) or kd2 (lane 8) Gprk2 mutants rescued the Smo phosphoshift in Gprk2-depleted cells, confirming that the Gprk2 mutants are catalytically inactive. (C) Rescue of ptc-luc reporter activity in gprk2-depleted S2-R+ cells. Treatment of cells with gprk2 5′- and 3′-UTR dsRNAs reduced Hh-dependent ptc-luc reporter activity, and this was rescued by re-expressing wild-type Gprk2. For this experiment, cells were cultured at the restrictive temperature for Gprk2 of 29°C.Gprk2 promotes target gene expression downstream of Smo in S2-R+ cells in a catalytic activity-dependent manner. (A) Rescue of Smo(TIF)Click here for additional data file.Figure S4SD-GFP variants from a ptc-luc reporter assay setup as in c1-4A-GFP in cells treated with or without Hh. Cell surface proteins were labeled by surface biotinylation. After lysis, biotinylated proteins were recovered by avidin-mediated affinity purification, and separated by SDS-PAGE. Smo was detected in the biotin-labeled surface protein fraction by immunoblotting with anti-GFP antibody. A background band served as a loading control to ensure that the starting samples had equivalent amounts of protein. Mutation of the Gprk2 phosphorylation sites did not impair the ability of Smo to reach the cell surface in response to Hh.Expression and cell surface accumulation of Gprk2 phosphosite cluster mutant forms of Smo. (A) Immunoblot analysis of Gprk2 phosphorylation cluster Ala mutant Smo(TIF)Click here for additional data file.Figure S5ptc-luc reporter activity driven by SmoSD variants with a subset of sites within clusters 1 or 2 mutated to Ala. Mutation of Ser604 and Thr606 (SmoSD.c1AATT) significantly reduced activity , but not as much as mutation of all four residues . Mutation of Ser604 and Thr606 individually (SmoSD.c1ATTT and SmoSD.c1SATT) had much less effect than mutating both. Mutation of both Thr610 and Thr612 in cluster 1 (SmoSD.c1STAA) had no significant effect on Smo activity. The situation was similar for cluster 2, where mutating just three residues in either half of the cluster (SmoSD.c2AAASST and SmoSD.c1SSTAAA) reduced activity , but both had less effect than mutating all six . (B) Stimulation of Hh-dependent ptc-luc activity by Gprk2 phosphocluster mutants in SmoWT backbone. Mutation all four cluster 1 or all six cluster 2 phosphorylation sites impairs Smo activity . Cluster 3 or 4 mutations have no significant effect.Multisite phosphorylation within Gprk2 phosphorylation clusters 1 and 2 is important for Smo activation. (A) (TIF)Click here for additional data file.Figure S6ptc-luc reporter assay setup as in Expression analysis of C-terminally truncated Smo variants. Immunoblot analysis of the indicated C-terminally truncated Smo-GFP proteins from a (TIF)Click here for additional data file.Figure S7ptc-luc reporter activity in Hh-treated S2 cells is significantly reduced by treatment with dsRNA targeting CKI (p<.001), confirming that CKI was depleted. (B) Western blot analysis of GFP immunoprecipitates from S2 cells expressing SmoSD-GFP and treated with control, gprk2, or ck1 dsRNA. The blot was probed with the Smo anti-pT610/pT612 phosphospecific antiserum (top) or with anti-Smo (bottom). Whereas Gprk2 depletion reduced bulk Smo phosphorylation and T610/T612 phosphorylation, CKI depletion had no discernible effect.CKI does not phosphorylate the four mapped Gprk2 phosphorylation site clusters. (A) (TIF)Click here for additional data file.Table S1SD peptides from LC-MS/MS analysis. Values represent area-under-the-curve measurements of precursor ion LC spectra (in arbitrary units).Quantification of phosphorylated and matching non-phosphorylated Smo(DOC)Click here for additional data file.


Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma. Neuroblastoma is a childhood cancer for which therapeutic progress has been slow. We analyzed a large number phosphorylated proteins in neuroblastoma cells to discern patterns that indicate functional signal transduction pathways. To analyze the data, we developed novel techniques that resolve data structure and visualize that structure as networks that represent both protein interactions and statistical relationships. We also fractionated neuroblastoma cells to examine the location of signaling proteins in different membrane fractions and organelles. The analysis revealed that signaling pathways are functionally and physically compartmentalized into distinct collaborative groups distinguished by phosphorylation patterns and intracellular localization. We found that two related proteins (FYN and LYN) act like central hubs in the tyrosine kinase signaling network that change intracellular localization and activity in response to activation of different receptors. Neuroblastoma arises from cells of the neural crest, a population of multipotent, migrating cells that differentiate into neurons in the peripheral nervous system, melanocytes, and structural cells . NeuroblTo address this clinically challenging problem, a greater understanding of the signaling mechanisms that are active in neural crest and neuroblastoma is required. Tyrosine kinase signaling networks play a major role in governing cell differentiation, including in neuroblastoma . There aTyrosine kinase signaling mechanisms are intimately intertwined with mechanisms that govern protein interactions in endocytosis. Src Homology 3 (SH3) domains are among the most abundant protein domain modules encoded by eukaryotic genomes; over 300 SH3 domains are found in 213 human proteins ,25. SH3 To identify patterns in tyrosine phosphorylation in neuroblastoma, we acquired phosphoproteomic data from 21 neuroblastoma cell lines and cell fractions including endosomes and detergent-resistant lipid rafts as previously characterized ,33. New To identify patterns in tyrosine phosphorylation in neuroblastoma, we analyzed tyrosine phosphoproteomic data acquired from 21 neuroblastoma cell lines using immunoprecipitation of tyrosine phosphorylated peptides as previously described ,42. FourWe experimented with different ways to analyze the mass spectrometry data . For the first analysis described below, phosphopeptide amounts were summed for each protein in each sample, with the exception of the SRC-family kinases (SFKs), where the C-terminal inhibitory phosphorylation was summed separately and given the names SRC_i; LYN_i; FYN_i; and YES1_i. This provided an overview of which proteins were present and phosphorylated together in the same samples. For the second analysis, phosphopeptides were summed into individual phosphorylation sites, which were then clustered. Clustering data were obtained by treating all samples mathematically as different states in the neuroblastoma system. We describe analysis of the whole dataset first, then subsets of the data, focusing on signaling proteins in endosomes and detergent-resistant membranes.1622 phosphorylated proteins were identified in all neuroblastoma samples . 1203 ofWe hypothesize that proteins containing tyrosine kinase, tyrosine phosphatase, SH2 and SH3 domains (PNCPs) will collectively initiate and control phosphotyrosine signaling pathways ,24. In nThese data indicate that neuroblastoma cell lines express and phosphorylate a large fraction of the PNCPs in the human genome. This remarkable diversity in phosphotyrosine signaling pathways likely represents a snapshot of signaling pathways activated in the sympathoadrenal lineage of neural crest that gives rise to neuroblastoma at different stages of development –6. The rS1 Dataset). Clusters were evaluated internally, based on the primary data, and externally, using PPI and gene ontology (GO) databases  embeddings were typically overlapping but not identical, yet reasonably close in their ability to resolve meaningful clusters as determined by external and internal evaluations  shown in An alternative visualization of data structure is a co-cluster correlation network CCCN; . In thiswithin these collaborative groups share a number of co-clustering downstream proteins in common, the only effector proteins in common between these two collaborative groups are PIK3R2, FYN, and the SFK scaffold protein, PAG1 . Ph. Ph32]. e.g., DDR2, ALK, KIT, RET, EGFR, PDGFA, FGFR1). FYN and LYN containing both activating and inhibiting phosphorylations were also prominent in endosomes, along with PAG1, inhibited SRC (SRC_i), the SH3 adaptor protein BCAR1, several other adaptor proteins, two tyrosine phosphatases (PTPN11/SHP-2 and PTPRN), and PLCG1/PLCγ1, which was found previously in endosomes in PC12 cells [C. elegans were found in neuroblastoma endosome fractions, and 2 of the 55 were detected in lysosome fractions [Endosomes from three neuroblastoma cell lines were characterized by phosphoproteomic analysis. In all endosome fractions from three cell lines ), 908 proteins were detected, including 22 RTKs, 10 tyrosine phosphatases; 30 SH2- and 44 SH3-domain-containing proteins. The most highly phosphorylated RTKs in neuroblastoma were those identified in 12 cells . Notablyractions .We asked whether particular phosphorylated proteins were enriched in endosomes and DRMs by calculating the ratio between amounts in those fractions compared to proteins in all other samples from the same cell line. ALK, FGFR1, RET, PDGFRA, DDR2, EGFR, and IGF1R were enriched in endosomes from two or more neuroblastoma cell lines, but there were profound differences among cell lines . In Fig We noted differences in distribution of SFK and PAG1 phosphorylation on individual phosphorylation sites between the two cell lines . For exaThese data suggest the hypothesis that stimulation of different RTKs should affect the activity and intracellular localization of FYN and LYN. We used a cell fractionation approach to assay intracellular localization after stimulation of ALK with PTN and KIT with SCF . AmountsThese data motivated further higher resolution interrogation of the relationships between individual protein phosphorylation events. We investigated the relationships among phosphorylation sites by clustering phosphorylation sites (summed from homologous phosphopeptides) and visualizing data structure as a co-cluster correlation network (CCCN). The edge-weighted, spring-embedded layout of this network showed several distinct groups of sites with statistical relationships to other groups . The datStrikingly, a number of phosphorylation sites on PAG1 were detected, but none were statistically clustered with activated FYN and inhibited LYN , while tThe data suggest that RTK phosphorylation does not occur on all sites at once under all conditions, resulting in different phosphorylation sites on ALK and other RTKs clustering separately from one another. RTKs phosphorylated on different sites also fractionated to endosomes and DRMs selectively . For exaThere is considerable interest in tyrosine kinase signaling mechanisms because of their roles in tumor initiation and metastasis. Tyrosine kinase signaling mechanisms arose during evolution when multicellular organisms evolved ,54, and Several technical hurdles had to be overcome when analyzing these data. 1) Phosphoproteomic data, like any mass spectrometry data, has missing values because many peptides are not analyzed by the detector, and using a “data not available” marker (NA) instead of zero facilitated calculation of statistical relationships based only on observed data . 2) EmplImportantly, to visualize data structure in a new and informative way, we developed an innovation that may be generally useful, namely to filter edges to show interactions only between co-clustered components CFN,  Figs. InThe observation that neuroblastoma cell lines expressed more than half of the RTKs in the human genome , and resCompartmentalization of signal-initiating receptors and downstream effectors may be employed to distinguish extracellular instructions that determine cell fate. Many receptors signal from endosomes to amplify signals, activate different effectors than those activated at the plasma membrane, or convey signals to different intracellular locations ,57–61. IDifferent RTKs elicit different cellular responses, yet all appear to activate the canonical RAS/ERK, PLC-γ, SFK, and PI3K/AKT pathways. Differential responses may be obtained by affecting the duration of downstream effector activation , or by mPrevious work supports the hypothesis that SFKs function to affect signal integration and protein localization. SFK family members are differentially palmitoylated, which affects their localization on endosomes and the plasma membrane ,70. SFKsThe transmembrane SFK scaffold protein PAG1 (Cbp/PAG) has been previously described to associate with lipid rafts . ConsistThe collaborative groups that emerged from these data  suggest SFKs may also play a role in RTK transactivation . SFK SH2Point mutations in the RTK, ALK, are the primary cause of familial neuroblastoma and account for 8–12% of sporadic neuroblastomas . ALK is Like ALK, KIT is an emerging marker for aggressive neuroblastoma that leads to poor prognosis . We founNTR enhances sensitivity to low neurotrophin levels, which affects response and outcome in NTRK1/2-expressing cells [Neurotrophin receptors are of interest in neuroblastoma because they are markers for clinical prognosis. NTRK1/TrkA is a marker for neuroblastoma tumors that spontaneously undergo apoptosis and regression, while NTRK2/TrkB is often expressed with its ligand, (BDNF), forming an autocrine loop that predicts poor prognosis –101. Theng cells . Overexpng cells . The difng cells ,104,105.Neuroblastoma cell lines offer insight into neural crest signaling pathways that is difficult to obtain directly from migrating immature neural crest cells. While signaling pathways activated by oncogenic mechanisms and cell culture conditions no doubt contribute to the phosphorylation patterns we identified here, the fact that these cells retained the capacity to migrate and differentiate  indicateDiscovery of new pathways activated in neuroblastoma cells provides potentially new therapeutic approaches . Co-acti3  and 10% fetal bovine serum .21 neuroblastoma cell lines were obtained by Cell Signaling Technology  from American Type Culture Collection ; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures ; Coriell Institute for Medical Research ; and Interlab Cell Line Collection . SMS-KCN, LAN6, SK-N-BE(2), and SH-SY5Y, were obtained by M.G. from Children’s Hospital of Los Angeles, CA, except for SH-SY5Y cells, which were provided by Dr. Mark Israel . Neuroblastoma cells were grown in RPMI 1640 medium  supplemented with NaHCOnnr5 cells . The γ-retroviral genomic vector plasmid (pRT43.2TrkCFP), helper plasmids (pIK6.1.gagpol+ATG and pLP-VSVG) were transfected into HEK293T cells using calcium phosphate. Cell culture media (Dulbecco’s Modified Eagle Medium/10% FBS) was changed approximately 16 hours post-transfection. Supernatant containing viral particles was harvested at 48 and 72 hours post-transfection and pooled together.Tyrosine phosphoproteomic data for 21 neuroblastoma cell lines were initially acquired at Cell Signaling Technology using techniques described previously ,42. CellCell lines were treated  with ligands or the ALK inhibitor TAE684 as indicated in For organelle fractionation phosphoproteomics, cell lines were treated with ligands (LAN-6 and TrkA-CFP-expressing SK-N-BE(2): NGF, SMS-KCN:BDNF) for 10 min at 37°C. For cell fractionation experiments after ALK and KIT stimulation , LAN-6 cAntibodies used in genenames.org). In cases where conserved peptide sequences identified multiple proteins, if a protein was identified by a different peptide in the sample, the peptide was assigned to that protein, otherwise the first name was used (this is referred to as exclusively summed). Where phosphorylation sites were known to have inhibitory effect on protein activity (Regulatory_sites.gz), peak intensity values were converted to negative values  ,42. Datae.g., duplicate runs of the same experiment), data were merged to include the average of the two runs, ignoring missing values. Otherwise, each experiment was treated as an independent sample for data analysis.In cases where duplicate mass spectrometry analyses were conducted on the same cell line, under the same conditions within a short time frame (S3 Dataset). Primary phosphoproteomics data are available from PhosphoSitePlus (http://www.phosphosite.org/browseDiseaseResultAction.do?id=66&type=true) using curation set (CS) numbers 1119, 1121, 1148, 1154, 1157, 1206, 1448, 1613, 1762, 1763, 1764, 1886, 1887, 1888, 2042, 2043, 2044, 3492, 3493, 3495, 4010, 4011, 5180, 5181, 5182, 5269, 5270, 5271, 5272, 6121, 6122, 6123, 6124, 6125, 6151, 6152, 6153, 6154, 6155, 9206, 9208, 9942, 9943, 9944, 9946, 10553, 10554, 10555, and 10557. Supplementary data, and links to spectra and other important experimental parameters from this submission will be also made available via the ‘Reprints, References, Supplemental Tables’ page http://www.phosphosite.org/staticSupp.do.Summarized data are available online as Supplemental Data ; LAN6.NGF ; SMSKCN.Control ; SMSKCN.NGF ; SMSKCN.Control ; SMSKCN.BDNF .1203 tyrosine phosphorylated and 557 AKT-substrate (using RxRxxS/T consensus sequence antibodies) proteins were identified in all samples; 138 were in common between phosphotyrosine and phospho-AKT substrate data.For analysis of proteins, the total amount of phosphorylation of each protein was determined by summing peak intensity signals from all peptides for each protein in each sample. In cases where conserved sequences did not allow unambiguous assignment to a particular protein, peptides were assigned to proteins that were detected by other phosphopeptides in the same sample or the first name was used. We thus obtained a data matrix in which each row corresponds to a protein and each column corresponds to a neuroblastoma cell line or organelle fraction  technique, t-distributed stochastic neighbor embedding (t-SNE) was employed to visualize the proteins in a scatter plot based on the distance or dissimilarity matrices ,48,123. rgl . FiltersClusters were evaluated by several quantitative measures. For comparison, 70 non-overlapping random clusters were generated containing gene names from the data set; the number of members was also randomized to mimic the number of genes identified in clusters defined by t-SNE embedding and minimum spanning tree methods. Evaluations based on examining the primary data  were performed as described . A quantstring.embl.de/) [genemania.org/) [phosphosite.org) [PPI edges from String (mbl.de/) , GeneMANia.org/) , and theite.org)  were merite.org) . Networkbioconductor.org/) using a p-value <0.01. If there was enrichment, at least two genes in the cluster should have the same GO term, so terms with single genes were discarded. The enriched GO terms per gene was compared to the average background for randomly selected genes from the dataset; this background was about one enriched GO term for every three genes [Gene Ontology (GO) was determined as described . Enricheee genes . When thee genes .For phosphorylation site analyses, peptide peak intensity values were summed based on sequence homology and phosphorylation site, independent of the presence or absence of oxidized methionine. In cases where conserved sequences did not allow unambiguous assignment to a particular protein, the peptide name either retained multiple names, for example “FYN 420; LCK 394; SRC 419; YES1 426,” or were merged into all possible larger peptides, for example MAPKs and C-terminal inhibitory phosphorylations on SRC, FYN, and YES1 (referred to as inclusively summed).Four neuroblastoma cell lines , SMS-KCN, and SY5Y) were cultured in 25 μL hanging drops containing approximately 5,000 neuroblastoma cells. Cells were transplanted into the neural crest of developing chick embryos to determine if these cells could survive transplantation and subsequently integrate into the migration pathways of the chick neural crest cells. Of the 14 embryos that were injected, 10 survived the transplantation process: three of these were injected with LAN6 cells, two with SK-N-BE(2) cells, three with SMS-KCN cells, and two with SY5Y cells. Fluorescent imaging of embryo sections showed that all four cell lines were successfully transplanted and could be located within various areas of the embryo with the use of GFP infection with adeno-associated virus that expresses GFP , anti-GFP , anti-ERGIC , and fluorescent secondary antibodies . Further details of embro transplantation methods are provided in S1 MoviePPI edges were filtered to show interactions only among proteins that cluster together. The movie was made from Cytoscape using RCytoscape to iteratively rotate and zoom the network nodes, plotted using coordinates derived from t-SNE SED embedding. Nodes and edges are graphed as in Figs (M4V)Click here for additional data file.S1 FigThe size and color of nodes is scaled to graph total phosphopeptides detected for each protein; blue represents phosphorylation on inhibitory sites, yellow, all other sites. Edge thickness represents a quantity (weight) that indicates the strength of evidence for interactions. There were 192 isolated nodes without edges in the network.(PDF)Click here for additional data file.S2 FigA) The neuroblastoma network obeyed the power law degree distribution typical of scale-free biological networks: α = 1.170; R2 = 0.795 for all degrees, α = 1.496, R2 = 0.820 for degrees > 10. The entire neuroblastoma phosphoproteomic network of 1622 proteins and 18728 interactions has a clustering coefficient of 0.167 and obeys the power law degree distribution typical of scale-free biological networks. This clustering coefficient, the network diameter of 7 (the longest length between connected nodes), and mean path length of 2.78, is consistent with the small-world effect, which is a property of real biological networks. Thus, the highly interconnected network of phosphorylated proteins in neuroblastoma indicates a robust biological network as opposed to a sparse or random selection of proteins [B) The most highly interconnected region of the neuroblastoma phosphoproteomic PPI network  is an almost perfect clique (a group where every node is connected to every other node). The group is made up of the SFKs , RTKs, EGFR, PDGFRB, KIT, other tyrosine kinases , a tyrosine phosphatase (SHP-2/PTPN11), and other tyrosine kinase signaling effector proteins that contain SH2 and/or SH3 domains. These 27 nodes are in turn connected to 711 nodes, or 44% of the total proteins in the neuroblastoma network shown in (proteins . (B) The(PDF)Click here for additional data file.S3 FigRows were sorted by hierarchical clustering using a modified distance function that can handle missing values.(PDF)Click here for additional data file.S4 FigA, top) GFP-expressing neuroblastoma cells, transplanted into chick embryos, express the neural crest marker HNK, and colonize derivatives ventral to the dorsal aorta as well as progenitor zones within the dorsal root ganglia (DRG) including the dorsal pole and lateral perimeter [A, bottom) Neuroblastoma cells give rise to afferents in the dorsal root and sympathetic ganglia that exhibit normal neuronal morphology  and colocalize with the neuronal marker Tuj-1. (B) Number of neuroblastoma cells according to their final migration location within the chick embryo and cell type. 164 LAN-6; 102 SK-N-BE(2); 86 SMS-KCN; and 142 SY5Y cells were detected in chick embryos after transplantation using human-specific anti-ER-Golgi intermediate compartment marker Click here for additional data file.S5 FigA), higher index (B), more edges per cluster (C), more edge weight per cluster (D), more GO term mean count over expected (E), and more GO terms per gene (F) than the random clusters. All graphs except A are plotted on a log scale. Statistical significance determined by the Welch two-sided t-test comparing random clusters to all t-SNE clusters is p < 0.0001 (A); p < 0.000001 (B); p < 0.00005 (C); p < 0.0002 (D); p < 0.00005 (E); and p < 0.006 (F).Clusters identified from Spearman, Euclidean, or SED t-SNE embeddings were validated by internal and external evaluations as described . Compare(PDF)Click here for additional data file.S6 FigA), PDGFRB (B), and KIT (C), graphed as PPI networks (left) and heat maps (right) as in A), PDGFRB and the Rho-GEF, intersectin 2 (ITSN2) and the adaptor, STAM2 (B), and the RTKs, KIT and ROR1 (C).Proteins that cluster in all three dissimilarity representations  with EPHA2 ((PDF)Click here for additional data file.S7 FigThis graph is similar to (PDF)Click here for additional data file.S8 FigA) and EGFR group (B), graphed as heat maps as in ALK group ((PDF)Click here for additional data file.S9 FigA), and other tyrosine kinases (B). Fold changes are graphed on a blue-yellow color heat map as in Shown are changes of more than twofold from representative experiments where peak intensity was measured for treatment and control conditions in the same experiment with cell lines and treatments indicated on column labels as in (PDF)Click here for additional data file.S10 FigFractionation strategy for cell fractions as performed previously ; 33. End(PDF)Click here for additional data file.S11 Figedge weight = e(20 * correlation) to cause the edge-weighted, spring-embedded layout to graph negatively correlated nodes far apart. Peptides were inclusively summed for this phosphorylation site network Click here for additional data file.S12 FigEnrichment of phosphorylation sites in endosome and DRM fractions was calculated as the ratio of amounts in endosomes or DRMs vs. the average in all other fractions and samples from that cell line, graphed as heat maps as in (PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.S3 Dataset(ZIP)Click here for additional data file.


Phosphorylation state of dopamine- and cAMP-regulated phosphoprotein, molecular weight 32kDa (DARPP32) is crucial to understand drug-mediated synaptic plasticity. In this study, mechanisms underlying repeated cocaine-stimulated phosphorylation of DARPP32 at threonine 75 (pDARPP32-Thr75) were determined by investigating the hypothesis that activation of protein kinases and phosphatases coupled to glutamate signaling is necessary for the regulation of pDARPP32-Thr75 after repeated cocaine administration.Intracaudate drug infusions into the rat dorsal striatum followed by Western immunoblot analysis were mainly performed to test this hypothesis.2+ concentrations followed by Ca2+-dependent protein kinase activation through stimulation of Ca2+ channels in striatal neurons were necessary for the phosphorylation. Activation of protein phosphatases further regulated the phosphorylation state by deactivating pDARPP32-Thr75 and upstream protein kinases.The results demonstrated that 7 repeated daily intraperitoneal injections of cocaine (20mg/kg) upregulated the expression of pDARPP32-Thr75. Increases in the cytosolic Ca2+-dependent manner.These findings suggest that activation of protein kinases and phosphatases coupled to glutamate receptors controls the phosphorylation state of DARPP32-Thr75 after repeated exposure to cocaine in the dorsal striatum in a Ca DARPP32Previous studies have shown that acute and repeated cocaine administration phosphorylates Thr34 and Thr75 of DARPP32 in mice through PKA and CDK5 activation, respectively . CocaineAdult male Sprague-Dawley rats (210–250g) were obtained from Hyo-Chang Science Co. . Rats were separated into pairs and habituated for 3 days in home cages. After acclimation, the rats were handled for 3 days. Food and water were provided ad libitum, and the rats were maintained on a 12 h light/ dark cycle throughout the experimental period. In addition, temperature and humidity were maintained at 21°C to 23°C and 45 to 55%, respectively. Experiments were performed in a quiet room to minimize stress to the animals. All animal use procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the provisions of the NIH Guide for the Care and Use of Laboratory Animals.2+ chelator, BAPTA-AM (50 nmol), the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK801 (2 nmol), the competitive NMDA receptor antagonist, DL-AP5 (2 nmol), the L-type voltage operated Ca2+ channel blocker, nifidipine (60 nmol), the selective P- and N-type calcium channel blocker, ω-Agatoxin IVA (Agatoxin IVA) , the Na+ channel blocker, tetradotoxin citrate (TTX) (1 pmol), the ryanodine Ca2+ channel blocker, dantrolene (20 nmol), the trisphosphate inositol (IP3)-sensitive Ca2+ channel blocker, xestosponin C (0.004 nmol), the potent Ca2+ and calmodulin protein kinase II (CaMKII) inhibitor, KN93 (10 nmol), the selective mitogen-activated protein kinase inhibitor for extracellular-regulated kinase 1/2 (ERK1/2), SL327 (150 nmol), the potent and selective protein kinase C (PKC) inhibitor, GF109203X (20 nmol), the selective c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (200 nmol), the potent and selective PKA inhibitor, KT5720 (5 nmol), the selective PP1/2A inhibitor, okadaic acid (0.5 nmol) or the potent PP2B inhibitor, cyclosporin A (0.5 nmol), was dissolved in the minimum concentration of dimethylsulfoxide (DMSO) and then diluted in artificial cerebro-spinal fluid (aCSF) containing (mM) 123 NaCl, 0.86 CaCl2, 3.0 KCl, 0.89 MgCl2, 0.50 NaH2PO4, and 0.25 Na2HPO4 aerated with 95% O2/5% CO2, pH 7.2 to 7.4 or NaCl. The same DMSO-aCSF mixture solution was used as the vehicle control for a given drug. The drug solutions were all adjusted to neutral with NaOH if necessary. Rats were received repeated intraperitoneal (i.p.) injections of saline or cocaine (20mg/kg) once per day for 7 consecutive days. Cocaine was injected in a volume of 1mL in physiological saline (0.9% sodium chloride). The concentrations of drugs were determined from previous studies , were purchased from Tocris Bioscience  and working solutions were always prepared fresh for experiments. The Ca studies  except f studies .Rats were anesthetized with 8% chloral hydrate  then placed in a stereotaxic apparatus. Under aseptic conditions, a 23-gauge stainless steel guide cannula  was implanted 1mm anterior to the bregma, 2.5mm right of the midline, and 5mm below the surface of the skull. The guide cannula was sealed with a stainless-steel wire of the same length. The rats were then allowed to recover from surgery for 5 days prior to the experiment. On the day of the experiment, the inner steel wire was replaced with a 30-gauge stainless steel injection cannula  that protruded 2.5mm beyond the guide cannula. Throughout the experiments, all drugs were infused unilaterally into the central part of the right dorsal striatum 5 minutes prior to the final i.p. injection of cocaine or saline in a volume of 1 μL at a rate of 0.2 μL/min in freely moving rats . The pro3VO4, 1 EDTA, and 1 EGTA, sonicated 3 times for 9 seconds each, and then incubated on ice for 1 hour. After sonicating, samples were centrifuged at 13000rpm for 30 minutes at 4°C. The pellet, which primarily contains nuclei and large debris, was discarded, and the supernatant was centrifuged again at 13000rpm for 30 minutes at 4°C. The concentration of the solubilized proteins in the supernatant fraction was determined based on the Bradford method using a Bio-Rad Protein Assay . The proteins in the supernatant were resolved using 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, after which the separated proteins were transferred to a nitrocellulose membrane. Next, the membrane was blocked with blocking buffer containing 5% skim milk in a mixture of Tris-buffered saline and Tween-20 (TBST), then washed 3 times for 10 minutes each with TBST. After washing, the membrane was probed with either a rabbit primary antiserum against pDARPP32-Thr 75 (1:500), pCaMKII (1:1000), pERK1/2 (1:2000), or pPKC (1:10000) or a mouse primary antiserum against pJNK (1:1000) for 18 hours at 4°C on a shaker. The membrane was washed again then incubated with an appropriate rabbit-  or mouse secondary antiserum  under the same conditions as for the primary antiserum for 1 hour at room temperature. Next, immunoreactive protein bands were detected on X-ray films using enhanced chemi-luminescence reagents . The samples were probed for unphosphorylated proteins after stripping the same membrane that was confirmed to contain phosphorylated protein. The same membrane was also probed for β-tubulin (1:2000) to normalize the blots. Both rabbit primary and secondary antibodies against unphosphorylated proteins were diluted to 1:1000. Immunoreactive protein bands visualized on films were semiquantified using an imaging digital camera and the NIH Image 1.62 software as previously described . Data were expressed as the mean ± SEM for each group (n = 3–4 per group). We first determined whether repeated cocaine administration alters the phosphorylation of DARPP32-Thr75 in the rat dorsal striatum. To accomplish this, we surveyed changes in pDARPP32-Thr75 immunoreactivity at 20 minutes after the final saline or cocaine injection, because the activity of protein kinases is known to peak in the rat dorsal striatum at this time point . The res2+ influx via stimulation of glutamate receptors , significantly decreased the elevation of pDARPP32-Thr75 immunoreactivity after repeated cocaine administration  and DL-AP5 (2 nmol), respectively, significantly decreased the elevation of pDARPP32-Thr75 immunoreactivity to the basal level after repeated cocaine administration , did not alter the repeated cocaine-induced increase in pDARPP32-Thr75 immunoreactivity , significantly decreased the elevation of pDARPP32-Thr75 immunoreactivity after repeated cocaine administration  in GABAergic neurons , significantly decreased the repeated cocaine-induced increase in pDARPP32-Thr75 immunoreactivity , did not (Stimulation of mGluRs potentiates diacylglycerol (DAG) and IP neurons , leadingactivity ; however did not .2+-dependent protein kinases and their interactions responsible for the phosphorylation of DARPP32-Thr75 were determined in this study. Intracaudate infusion of the CaMKII inhibitor, KN93 (10 nmol), significantly decreased the repeated cocaine-induced increase in pDARPP32-Thr75 immunoreactivity , significantly increased the repeated cocaine-induced increase in pDARPP32-Thr75 immunoreactivity . Similar2+ concentrations followed by protein kinase activation through both NMDA receptors and ryanodine Ca2+ channels were found to be required for the phosphorylation. Moreover, activation of PPs further regulated the phosphorylation state of DARPP32-Thr75 by dephosphorylating it and downstream protein kinases. These findings suggest that glutamate receptor-induced activation of protein kinases and PPs is crucial for the regulation of pDARPP32-Thr75 after repeated cocaine exposure.DARPP32 is thought to be a key molecule in drug-induced synaptic plasticity that functions by integrating dopaminergic and glutamatergic transmission in the basal forebrain . In the 2+ influx in the present study, suggesting that activation of Ca2+ channels is required for the phosphorylation. Blockade of NMDA receptors, but not L-, N-, and P-type Ca2+ channels, abolishes the repeated cocaine-induced increase in pDARPP32-Thr75, indicating that NMDA receptors may act as primary Ca2+ channels for the phosphorylation. However, the possibility that other Ca2+ channels can contribute to the phosphorylation at other time points should not be excluded because L-type Ca2+ channels have the capability to produce Ca2+ cascades in a very short-time period in GABAergic neurons /CDK5 in mice (3 is more effective to evoke the phosphorylation. In addition to CDK5, PKC can be activated by activation of both NMDA receptors and mGluRs via increases in Ca2+ influx and PI hydrolysis, respectively (2+ mobilization, which is likely necessary for the phosphorylation of DARPP32-Thr75 in response to repeated cocaine exposure.Blockade of Nastriatum . This inl stores . Taken tn the ER , leading in mice . These fectively . Thus, a2+ concentrations activate Ca2+-dependent protein kinases (2+ followed by activation of protein kinases is responsible for the phosphorylation of DARPP32-Thr75 after repeated cocaine administration. Interactions of protein kinases in turn regulate the phosphorylation state of DARPP32-Thr75 in a complicated manner. In the present study, inhibition of CaMKII following repeated cocaine administration was found to reduce ERK1/2, but not PKC and JNK phosphorylation. Inhibition of PKC reduces ERK1/2 and JNK, but not CaMKII, phosphorylation. However, inhibition of either JNK or ERK1/2 does not influence any other protein kinases. These findings suggest that increases in cytosolic Ca2+ mobilization after glutamate receptor stimulation are crucial for phosphorylation of DARPP32-Thr75 through the activation of PKC/JNK, PKC/ERK1/2, and CaMKII/ERK1/2. As an upstream transducer of pDARPP32-Thr75, ERK1/2 can be phosphorylated by the stimulation of dopamine D1 receptors after repeated cocaine administration (Increases in cytosolic Ca kinases , which sstration . Stimulastration . Activatstration .Activation of PPs controls the phosphorylation state of signaling molecules after cocaine administration . In this2+ mobilization and leads to the phosphorylation of DARPP32-Thr75 in the rat dorsal striatum. As postulated in 2+ as well as Na+ influxes into the GABAergic neurons (2+ from the ER. This increase potentiates the activation of Ca2+-dependent protein kinases including PKC, CaMKII, ERK1/2, JNK, and upstream regulators of PKC. Protein kinase interactions such as PKC/JNK, PKC/ERK1/2, and CaMKII/ERK1/2 may result in DARPP32-Thr75 phosphorylation, probably via downstream intermediates affected by JNK and ERK1/2. In addition, PPs activated by the stimulation of glutamate receptors further regulate the phosphorylation state by dephosphorylating pDARPP32-Thr75 and protein kinases. Taken together, dynamic control of the phosphorylation state of DARPP32-Thr75 by protein kinases and phosphatases seems to be a key physiological event in cocaine-induced synaptic plasticity in the dorsal striatum.Repeated cocaine increases Ca neurons . IncreasNone.