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1.  A Global Protein Kinase and Phosphatase Interaction Network in Yeast 
Science (New York, N.Y.)  2010;328(5981):1043-1046.
The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses.
doi:10.1126/science.1176495
PMCID: PMC3983991  PMID: 20489023
2.  The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data 
Nature methods  2013;10(8):730-736.
Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org.
doi:10.1038/nmeth.2557
PMCID: PMC3773500  PMID: 23921808
3.  Tony Pawson OC OOnt CH FRS FRSC 
Current Oncology  2013;20(5):e359.
doi:10.3747/co.20.1754
PMCID: PMC3805405
4.  Modular evolution of phosphorylation-based signalling systems 
Phosphorylation sites are formed by protein kinases (‘writers’), frequently exert their effects following recognition by phospho-binding proteins (‘readers’) and are removed by protein phosphatases (‘erasers’). This writer–reader–eraser toolkit allows phosphorylation events to control a broad range of regulatory processes, and has been pivotal in the evolution of new functions required for the development of multi-cellular animals. The proteins that comprise this system of protein kinases, phospho-binding targets and phosphatases are typically modular in organization, in the sense that they are composed of multiple globular domains and smaller peptide motifs with binding or catalytic properties. The linkage of these binding and catalytic modules in new ways through genetic recombination, and the selection of particular domain combinations, has promoted the evolution of novel, biologically useful processes. Conversely, the joining of domains in aberrant combinations can subvert cell signalling and be causative in diseases such as cancer. Major inventions such as phosphotyrosine (pTyr)-mediated signalling that flourished in the first multi-cellular animals and their immediate predecessors resulted from stepwise evolutionary progression. This involved changes in the binding properties of interaction domains such as SH2 and their linkage to new domain types, and alterations in the catalytic specificities of kinases and phosphatases. This review will focus on the modular aspects of signalling networks and the mechanism by which they may have evolved.
doi:10.1098/rstb.2012.0106
PMCID: PMC3415845  PMID: 22889906
protein kinase; protein phosphatase; interaction domain; phosphorylation; domain linkage; coevolution
5.  Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response 
Cancer research  2010;70(20):7776-7787.
Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4+ T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell–deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2+ and basal breast cancers. We reveal a novel role for tumor cell–derived ShcA in the establishment and maintenance of an immunosuppressive state.
doi:10.1158/0008-5472.CAN-10-2229
PMCID: PMC3660232  PMID: 20924104
6.  A Web Resource for Improved Analysis of AP-MS Protein Interaction Data 
Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, since negative controls are largely bait-independent, we reasoned that the negative controls generated by the proteomics research community could be developed as a resource for scoring AP-MS data.
Here we present the Contaminant Repository for Affinity Purification (The CRAPome), currently containing AP-MS data from 343 control purifications conducted by 11 different research groups (www.crapome.org). Users employ an intuitive graphical user interface to explore the database, by either querying one protein at a time, downloading background contaminant lists for selected experimental conditions, or uploading their own data (alongside their own negative controls when available) and performing data analysis. The CRAPome database scores contaminants vs. true interactors based on semi-quantitative mass spectrometry data (normalized spectral counts) embedded in most mass spectrometry experiments. The Significance Analysis of INTeractome (SAINT) scoring scheme, in addition to a simpler Fold Change calculation (FC score) are used to score user-supplied data and return a ranked list of putative interactors. We also describe database structure and composition, provide examples of the use of this resource to filter contaminants with properly chosen controls, and demonstrate the utility of the scoring scheme for identifying bona fide interaction partners. The CRAPome accommodates a variety of purification schemes and, while currently focused on human data, will be expanded to other species.
PMCID: PMC3635329
7.  Construction of human activity-based phosphorylation networks 
A high-resolution map of human phosphorylation networks was constructed by integrating experimentally determined kinase-substrate relationships with other resources, such as in vivo phosphorylation sites.
High-quality kinase-substrate relationships (KSRs) were determined using an integrated approach that combines protein microarray technology and bioinformatics analysis.Phosphorylation motifs were predicted for 284 human kinases, representing 55% of the human kinome.A high-resolution map of human phosphorylation networks was constructed that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates.A new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling was discovered based on KSRs identified in the phosphorylation networks.
The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.
doi:10.1038/msb.2013.12
PMCID: PMC3658267  PMID: 23549483
phosphorylation; signaling networks; systems biology
8.  Yap- and Cdc42-Dependent Nephrogenesis and Morphogenesis during Mouse Kidney Development 
PLoS Genetics  2013;9(3):e1003380.
Yap is a transcriptional co-activator that regulates cell proliferation and apoptosis downstream of the Hippo kinase pathway. We investigated Yap function during mouse kidney development using a conditional knockout strategy that specifically inactivated Yap within the nephrogenic lineage. We found that Yap is essential for nephron induction and morphogenesis, surprisingly, in a manner independent of regulation of cell proliferation and apoptosis. We used microarray analysis to identify a suite of novel Yap-dependent genes that function during nephron formation and have been implicated in morphogenesis. Previous in vitro studies have indicated that Yap can respond to mechanical stresses in cultured cells downstream of the small GTPases RhoA. We find that tissue-specific inactivation of the Rho GTPase Cdc42 causes a severe defect in nephrogenesis that strikingly phenocopies loss of Yap. Ablation of Cdc42 decreases nuclear localization of Yap, leading to a reduction of Yap-dependent gene expression. We propose that Yap responds to Cdc42-dependent signals in nephron progenitor cells to activate a genetic program required to shape the functioning nephron.
Author Summary
The mammalian kidney undergoes reiterative and stereotypical morphogenetic changes to create the elaborately convoluted adult nephron, the functional filtration unit of the kidney. How these sequential morphological events are controlled remains poorly understood. Here we show that the transcriptional activator Yap is essential in the developing murine kidney. Yap mutants have reduced nephrogenesis and defective morphogenesis. Yap function in nephrogenesis is independent of its previously described role in regulation of cell proliferation and apoptosis. Instead, Yap activity is needed for proper expression of a suite of genes that control cell signaling and cell structure. Remarkably, we find that ablation of Cdc42 phenocopies loss of Yap. We show that Cdc42 is essential for nuclear access of Yap, both in vivo and in tissue culture studies. Taken together, our work shows that Yap and Cdc42 are essential for the cell fate and morphogenesis decisions necessary to shape functioning nephrons, and suggests that Yap functions downstream of Cdc42 during kidney development.
doi:10.1371/journal.pgen.1003380
PMCID: PMC3605093  PMID: 23555292
9.  MERTK Interactions with SH2-Domain Proteins in the Retinal Pigment Epithelium 
PLoS ONE  2013;8(2):e53964.
The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK571–999), purified and phosphorylated. Ni2+-NTA pull downs were performed using 6xHis-rMERTK571–999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK571–999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.
doi:10.1371/journal.pone.0053964
PMCID: PMC3563642  PMID: 23390493
10.  Splice-Mediated Motif Switching Regulates Disabled-1 Phosphorylation and SH2 Domain Interactions 
Molecular and Cellular Biology  2012;32(14):2794-2808.
Disabled-1 (Dab1) plays a key role in reelin-mediated neuronal migration during brain development. Tyrosine phosphorylation of Dab1 at two YQXI and two YXVP motifs recruits multiple SH2 domains, resulting in activation of a wide range of signaling cascades. However, the molecular mechanisms underlying the coordinated regulation of Dab1 downstream effectors remain poorly understood. Here, we show that alternative splicing results in inclusion of different combinations of YQXI and YXVP motifs in Dab1 isoforms during development. Dab1 variants with partial or complete loss of YQXI motifs are preferentially expressed at early developmental stages, whereas the commonly studied Dab1 is predominantly expressed at late developmental stages. Expression of Dab1 variants in 293T and Neuro2a cells reveals reduced levels or absence of tyrosine phosphorylation in variants that have lost one or both YQXI motifs. We further demonstrate that Dab1 variants differ in their abilities to activate Src and recruit distinct SH2 domains involved in specific downstream signaling pathways. We propose that coordinated expression of specific Dab1 isoforms in different populations of cells in the developing brain contributes to precise neuronal migration by modulating the activity of subsets of Dab1 downstream effectors.
doi:10.1128/MCB.00570-12
PMCID: PMC3416185  PMID: 22586277
11.  Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54) 
Molecular and Cellular Biology  2012;32(13):2440-2453.
Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner.
doi:10.1128/MCB.06620-11
PMCID: PMC3434478  PMID: 22527282
12.  Gravin Is a Transitory Effector of Polo-like Kinase 1 during Cell Division 
Molecular cell  2012;48(4):547-559.
SUMMARY
The mitogenic and second-messenger signals that promote cell proliferation often proceed through multienzyme complexes. The kinase-anchoring protein Gravin integrates cAMP and calcium/phospholipid signals at the plasma membrane by sequestering protein kinases A and C with G protein-coupled receptors. In this report we define a role for Gravin as a temporal organizer of phosphorylation-dependent protein-protein interactions during mitosis. Mass spectrometry, molecular, and cellular approaches show that CDK1/Cyclin B1 phosphorylates Gravin on threonine 766 to prime the recruitment of the polo-like kinase Plk1 at defined phases of mitosis. Fluorescent live-cell imaging reveals that cells depleted of Gravin exhibit mitotic defects that include protracted prometaphase and misalignment of chromosomes. Moreover, a Gravin T766A phosphosite mutant that is unable to interact with Plk1 negatively impacts cell proliferation. In situ detection of phospho-T766 Gravin in biopsy sections of human glioblastomas suggests that this phosphorylation event might identify malignant neoplasms.
doi:10.1016/j.molcel.2012.09.002
PMCID: PMC3513578  PMID: 23063527
13.  The Adaptor Protein Grb2 Is Not Essential for the Establishment of the Glomerular Filtration Barrier 
PLoS ONE  2012;7(11):e50996.
The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.
doi:10.1371/journal.pone.0050996
PMCID: PMC3511449  PMID: 23226445
14.  Preface 
doi:10.1098/rstb.2012.0374
PMCID: PMC3415847  PMID: 22889902
15.  Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors 
The Journal of Cell Biology  2011;195(6):1033-1045.
Beyond homotypic receptor interactions that are required for Eph signaling, ligand-independent association and crosstalk between members of the EphA and -B subclasses determine cell signaling outcomes.
Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph–Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.
doi:10.1083/jcb.201104037
PMCID: PMC3241718  PMID: 22144690
16.  Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family 
Cell  2012;149(1):214-231.
Summary
Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.
Graphical Abstract
Highlights
► Human bromodomain family characterized with 29 high-resolution crystal structures ► Peptide arrays establish core histone binding preferences of BRD ► Interactions with histone-acetylated lysine sites are quantified ► Flanking posttranslational modifications greatly impact acetylated lysine recognition
Bromodomains bind acetylated lysines in histones, a key event in the “reading” of epigenetic marks. A foundation for designing inhibitors for this emerging class of drug targets is provided by a systematic biochemical analysis of their binding preferences and a compendium of 29 crystal structures of human bromodomains.
doi:10.1016/j.cell.2012.02.013
PMCID: PMC3326523  PMID: 22464331
17.  A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts 
Skeletal Muscle  2012;2:5.
Background
The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.
Methods
To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.
Results
Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.
Conclusion
Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.
doi:10.1186/2044-5040-2-5
PMCID: PMC3350448  PMID: 22394512
differentiation; FSBA; kinase assay; mitogen-activated protein kinase; myoblast; p38; phosphorylation; quantitative MS
18.  The ShcA Phosphotyrosine Docking Protein Uses Distinct Mechanisms to Regulate Myocyte and Global Heart Function 
Circulation research  2010;108(2):184-193.
Rationale
Although tyrosine kinases (TKs) are important for cardiac function, their relevant downstream targets in the adult heart are unknown. The ShcA docking protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal pTyr-binding (PTB) and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. Therefore, ShcA could be a potential hub for downstream TK signaling in the myocardium.
Objective
To define the role of ShcA, a TK scaffold, in the adult heart using a myocardial-specific knockout of murine ShcA (ShcA CKO) and domain knock-in models.
Methods and Results
ShcA CKO mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered collagen and extracellular matrix compliance properties, suggesting disruption of mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA CKO mice, whereas disruption of the SH2 domain caused a less severe cardiac phenotype. Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function but necessary to prevent adverse remodeling after hemodynamic overload.
Conclusions
These data demonstrate a requirement for TK-ShcA PTB domain signaling to maintain cardiac function. In addition, analysis of the SH2 domain and CH1 pTyr sites reveals that ShcA mediates pTyr signaling in the adult heart through multiple distinct signaling elements that control myocardial functions and response to stresses.
doi:10.1161/CIRCRESAHA.110.233924
PMCID: PMC3109628  PMID: 21148430
adaptor protein; tyrosine kinase; cardiomyopathy; signal transduction
19.  Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis 
Cell  2011;147(2):306-319.
SUMMARY
Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.
doi:10.1016/j.cell.2011.08.046
PMCID: PMC3202669  PMID: 22000011
20.  Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis 
Cell  2011;147(2):306-319.
Summary
Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.
PaperFlick
Graphical Abstract
Highlights
► The SH2-kinase domain interface is necessary for high catalytic activity of Bcr-Abl ► This intramolecular interaction is critical for Bcr-Abl-dependent leukemogenesis ► Disrupting this interaction potentiates the effects of clinical kinase inhibitors ► Targeting of the SH2-kinase interface with a monobody inhibits Bcr-Abl allosterically
Intramolecular interaction between two domains of Bcr-Abl is essential for its oncogenic activity. Disrupting the interaction prevents leukemia, even in cases where Bcr-Abl has become resistant to existing kinase inhibitors.
doi:10.1016/j.cell.2011.08.046
PMCID: PMC3202669  PMID: 22000011
21.  Mapping protein interactions by combining antibody affinity maturation and mass spectrometry 
Analytical Biochemistry  2011;417(1):25-35.
Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (VH) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 108 to 109 new variable light (VL) genes. Improved binders were isolated by stringent selections from these new “chain-shuffled” libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein–protein interaction networks.
doi:10.1016/j.ab.2011.05.005
PMCID: PMC3171153  PMID: 21704603
Antibody phage display; Affinity maturation; Immunoprecipitation; Mass spectrometry; Protein–protein interaction networks
23.  Positive Selection of Tyrosine Loss in Metazoan Evolution 
Science (New York, N.Y.)  2009;325(5948):1686-1688.
John Nash showed that within a complex system individuals are best off if they make the best decision that they can, taking into account the decisions of the other individuals. Here, we investigate if similar principles influence the evolution of signaling networks in multicellular animals. Specifically, by analyzing a set of metazoan species, we observe a striking negative correlation of genomically encoded tyrosine content with biological complexity (as measured by the number of cell types in each organism). We discuss how this observed tyrosine loss correlates with the expansion of tyrosine kinases in the evolution of the metazoan lineage and how it may relate to the optimization of signaling systems in multi-cellular animals. We propose that this phenomenon illustrates genome-wide adaptive evolution to accommodate beneficial genetic perturbation.
doi:10.1126/science.1174301
PMCID: PMC3066034  PMID: 19589966
24.  Phosphotyrosine Signaling: Evolving a New Cellular Communication System 
Cell  2010;142(5):661-667.
Tyrosine phosphorylation controls many cellular functions. Yet the three-part toolkit that regulates phosphotyrosine signaling -- tyrosine kinases, phosphotyrosine phosphatases, and Src Homology 2 (SH2) domains -- is a relatively new innovation. Genomic analyses reveal how this revolutionary signaling system may have originated and why it rapidly became critical to metazoans.
doi:10.1016/j.cell.2010.08.023
PMCID: PMC2950826  PMID: 20813250
25.  AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade 
Nature cell biology  2010;12(12):1242-1249.
Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities1. Processive relay of signals through RAF–MEK–ERK modulates cell growth and proliferation2,3. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases4,5. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP6,7. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838 on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events.
doi:10.1038/ncb2130
PMCID: PMC3042953  PMID: 21102438

Results 1-25 (66)