Brian A. Joughin

Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate: Role in dimer formation and ligand binding

ABSTRACT Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3ζ. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3ζ in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3ζ at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3ζ to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3ζ dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3ζ functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

Spatial Exclusivity Combined with Positive and Negative Selection of Phosphorylation Motifs Is the Basis for Context-Dependent Mitotic Signaling

Mitotic kinases that localize to the same compartment show negative selectivity for residues in each other’s recognition motifs. Motifs and Antimotifs Mitosis is a complex process controlled by multiple kinases, such as the cyclin-dependent kinase Cdk1/cyclin B, the Aurora kinases Aurora A and B, the kinase Nek2, and the Polo-like kinases, especially Plk1 (Polo-like kinase 1). Alexander et al. used in vitro assays and a peptide library screening method to refine the phosphorylation site selectivity of each of these kinases and found that those that were located in similar compartments within the cell during mitosis showed a strong negative selection for residues that were positively selected by other kinases in the same compartment. In contrast, those that were located in distinct compartments tended to recognize similar phosphorylation site motifs. These results led the authors to propose that mitotic kinase selectivity is determined by two factors—spatial exclusivity and “antimotif” specificity. The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.

PTMScout, a Web Resource for Analysis of High Throughput Post-translational Proteomics Studies

The rate of discovery of post-translational modification (PTM) sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. To help meet this challenge, we have created PTMScout, a web-based interface for viewing, manipulating, and analyzing high throughput experimental measurements of PTMs in an effort to facilitate biological understanding of protein modifications in signaling networks. PTMScout is constructed around a custom database of PTM experiments and contains information from external protein and post-translational resources, including gene ontology annotations, Pfam domains, and Scansite predictions of kinase and phosphopeptide binding domain interactions. PTMScout functionality comprises data set comparison tools, data set summary views, and tools for protein assignments of peptides identified by mass spectrometry. Analysis tools in PTMScout focus on informed subset selection via common criteria and on automated hypothesis generation through subset labeling derived from identification of statistically significant enrichment of other annotations in the experiment. Subset selection can be applied through the PTMScout flexible query interface available for quantitative data measurements and data annotations as well as an interface for importing data set groupings by external means, such as unsupervised learning. We exemplify the various functions of PTMScout in application to data sets that contain relative quantitative measurements as well as data sets lacking quantitative measurements, producing a set of interesting biological hypotheses. PTMScout is designed to be a widely accessible tool, enabling generation of multiple types of biological hypotheses from high throughput PTM experiments and advancing functional assignment of novel PTM sites. PTMScout is available at http://ptmscout.mit.edu.

A computational method for the analysis and prediction of protein:phosphopeptide‐binding sites

Phosphopeptide‐binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo‐box domains, as well as the 14‐3‐3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide‐binding domains was therefore developed to elucidate the physical and chemical nature of phosphopeptide binding, given this lack of structural similarity. The surfaces of nine phosphopeptide‐binding proteins, representing seven distinct classes of phosphopeptide‐binding modules, were discretized, and encoded with information about amino acid identity, surface curvature, and electrostatic potential at every point on the surface in order to identify local surface properties enriched in phosphoresidue contact sites. Cross‐validation indicated that propensities corresponding to this enrichment calculated from a subset of the training data could be used to predict the phosphoresidue contact site on proteins not used in training with no false negative results, and with few unconfirmed positive predictions. The locations of phosphoresidue contact sites were then predicted on the surfaces of the checkpoint kinase Chk1 and the BRCA1 BRCT repeat domain, and these predictions are consistent with recent experimental evidence.

Action-at-a-distance interactions enhance protein binding affinity

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen‐bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 β‐lactamase (TEM1) to the β‐lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1‐binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

The actin-binding protein Mena regulates RTK signaling after growth factor stimulation in tumor cells by a novel mechanism. The alternatively spliced MenaINV isoform disrupts this attenuation to drive sensitivity to growth factors, resistance to targeted inhibitors, and ultimately tumor invasion and metastasis.

DNA repair capacity in multiple pathways predicts chemoresistance in glioblastoma multiforme

Cancer cells can resist the effects of DNA-damaging therapeutic agents via utilization of DNA repair pathways, suggesting that DNA repair capacity (DRC) measurements in cancer cells could be used to identify patients most likely to respond to treatment. However, the limitations of available technologies have so far precluded adoption of this approach in the clinic. We recently developed fluorescence-based multiplexed host cell reactivation (FM-HCR) assays to measure DRC in multiple pathways. Here we apply a mathematical model that uses DRC in multiple pathways to predict cellular resistance to killing by DNA-damaging agents. This model, developed using FM-HCR and drug sensitivity measurements in 24 human lymphoblastoid cell lines, was applied to a panel of 12 patient-derived xenograft (PDX) models of glioblastoma to predict glioblastoma response to treatment with the chemotherapeutic DNA-damaging agent temozolomide. This work showed that, in addition to changes in O6-methylguanine DNA methyltransferase (MGMT) activity, small changes in mismatch repair (MMR), nucleotide excision repair (NER), and homologous recombination (HR) capacity contributed to acquired temozolomide resistance in PDX models and led to reduced relative survival prolongation following temozolomide treatment of orthotopic mouse models in vivo Our data indicate that measuring the combined status of MMR, HR, NER, and MGMT provided a more robust prediction of temozolomide resistance than assessments of MGMT activity alone. Cancer Res; 77(1); 198-206. ©2016 AACR.

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

Phosphorylation of a tyrosine in the disordered N-terminal region of ACTN4 functions as a switch exposing a second site for phosphorylation. Flipping the phosphorylation switch How does phosphorylation of a protein at one site regulate phosphorylation of a second site? Travers et al. identified distinct roles for tandem phosphorylation sites in an intrinsically disordered region of α-actinin-4 (ACTN4). Molecular dynamics simulations, validated by experimental observations, indicated that phosphorylation on Tyr4 increased the accessibility of Tyr31 and thus phosphorylation of this site, which reduced ACTN4 binding to actin. Thus, the first site functioned as a switch that enabled phosphorylation at the second site, which controlled binding to actin. Tandem-site phosphorylation may be a mechanism by which spatiotemporal regulation of protein function evolved. The kinase for the first site may only be present or active at restricted locations or times, whereas the kinase for the second site may be constitutively active or ubiquitous, may have a loose consensus motif, or may be the same as the kinase for the first site, but has different kinetics for the second site. Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human α-actinin-4 (ACTN4), Tyr4 and Tyr31 are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr4 site. We found that introducing a phosphomimetic mutation at only Tyr31 was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr31 is mostly buried and that phosphorylation of Tyr4 would increase the solvent exposure and thus kinase accessibility of Tyr31. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr4, whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr4 and Tyr31. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr31 can be dictated by the kinase affinity for Tyr4. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site.

Protein kinases display minimal interpositional dependence on substrate sequence: potential implications for the evolution of signalling networks.

Characterization of in vitro substrates of protein kinases by peptide library screening provides a wealth of information on the substrate specificity of kinases for amino acids at particular positions relative to the site of phosphorylation, but provides no information concerning interdependence among positions. High-throughput techniques have recently made it feasible to identify large numbers of in vivo kinase substrates. We used data from experiments on the kinases ATM/ATR and CDK1, and curated CK2 substrates to evaluate the prevalence of interactions between substrate positions within a motif and the utility of these interactions in predicting kinase substrates. Among these data, evidence of interpositional sequence dependencies is strikingly rare, and what dependency exists does little to aid in the prediction of novel kinase substrates. Significant increases in the ability of models to predict kinase–substrate specificity beyond position-independent models must come largely from inclusion of elements of biological and cellular context, rather than further analysis of substrate sequences alone. Our results suggest that, evolutionarily, kinase substrate fitness exists in a smooth energetic landscape. Taken with results from others indicating that phosphopeptide-binding domains do exhibit interpositional dependence, our data suggest that incorporation of new substrate molecules into phospho-signalling networks may be rate-limited by the evolution of suitability for binding by phosphopeptide-binding domains.