Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Temporal regulation of EGF signalling networks by the scaffold protein Shc1.

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

Myotubularin-related proteins 3 and 4 interact with polo-like kinase 1 and centrosomal protein of 55 kDa to ensure proper abscission.

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.

The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

Affinity purification coupled with mass spectrometry (AP-MS) is 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 (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. 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 its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Localization of phosphorylated CK2alpha to the mitotic spindle requires the peptidyl-prolyl isomerase Pin1.

CK2 is a serine/threonine kinase with many substrates, largely unknown modes of regulation and essential roles in mitotic progression. CK2α, a catalytic subunit of CK2, is phosphorylated in mitosis, and here we examine the effect of phosphorylation on CK2α localization. Using phosphospecific antibodies, we show that CK2α localizes to the mitotic spindle in a phosphorylation-dependent manner. Mitotic spindle localization requires the unique C-terminus of CK2α, and involves a novel regulatory mechanism in which phosphorylation of CK2α facilitates binding to the peptidyl-prolyl isomerase Pin1, which is required for CK2α mitotic spindle localization. This could explain how the constitutive activity of CK2α might be targeted towards mitotic substrates. Furthermore, because Pin1 has many important spindle substrates, this might represent a general mechanism for localization of mitotic signalling proteins.

Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAß1. We show that unlike canonical cytosolic calcineurin, CNAß1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAß1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAß1.

Endofin is required for HD-PTP and ESCRT-0 interdependent endosomal sorting of ubiquitinated transmembrane cargoes.

Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

Phenotypic and Interaction Profiling of the Human Phosphatases Identifies Diverse Mitotic Regulators.

Reversible phosphorylation is a fundamental regulatory mechanism, intricately coordinated by kinases and phosphatases, two classes of enzymes widely disrupted in human disease. To better understand the functions of the relatively understudied phosphatases, we have used complementary affinity purification and proximity-based interaction proteomics approaches to generate a physical interactome for 140 human proteins harboring phosphatase catalytic domains. We identified 1,335 high-confidence interactions (1,104 previously unreported), implicating these phosphatases in the regulation of a variety of cellular processes. Systematic phenotypic profiling of phosphatase catalytic and regulatory subunits revealed that phosphatases from every evolutionary family impinge on mitosis. Using clues from the interactome, we have uncovered unsuspected roles for DUSP19 in mitotic exit, CDC14A in regulating microtubule integrity, PTPRF in mitotic retraction fiber integrity, and DUSP23 in centriole duplication. The functional phosphatase interactome further provides a rich resource for ascribing functions for this important class of enzymes.

Systematic investigation of hierarchical phosphorylation by protein kinase CK2.

Although multiple phosphorylation sites are often clustered in substrates, the mechanism of phosphorylation within clusters has not been systematically investigated. Intriguingly, in addition to acidic residues, protein kinase CK2 can use phosphoserine residues as consensus determinants suggesting that CK2 may act in concert with other kinases. We used a peptide array approach to outline optimal consensus sequences for hierarchical phosphorylation by CK2, both in the context of processive, multisite phosphorylation, and in concert with a priming proline-directed kinase. Results suggest that hierarchical phosphorylation involving CK2 requires precise positioning of either multiple phosphodeterminant residues or specific combinations of canonical determinants and phosphodeterminants, and can be as enzymatically favorable as canonical CK2 phosphorylation. Over 1600 human proteins contain at least one CK2 hierarchical consensus motif, and ~20% of these motifs contain at least one reported in vivo phosphorylation site. These motifs occur non-randomly in the human proteome, with significant enrichment in proteins controlling specific cellular processes. Taken together, our results provide strong in vitro evidence that hierarchical phosphorylation may contribute to the regulation of crucial biological processes. In addition, the results suggest a mechanism by which CK2, a constitutively active kinase, can be a regulatory participant in cellular processes. BIOLOGICAL SIGNIFICANCE: Phosphorylation is a crucial regulatory mechanism governing cellular signal transduction pathways, and despite the large number of identified sites to date, most mechanistic studies remain focused on individual phosphorylation sites. This study is the first to systematically determine specific consensus sequences for hierarchical phosphorylation events. The results indicate that individual phosphorylation sites should not be studied in isolation, and that larger, multisite phosphorylation motifs may have profound impact on cellular signaling. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Mass spectrometric tools for systematic analysis of protein phosphorylation.

The study of protein phosphorylation has long been performed one protein and one modification site at a time, with a major emphasis placed on the functional relevance of one or a small number of given phosphorylation event(s). This has enabled exquisitely detailed views of many intracellular signaling pathways but has left a large portion of the phosphoproteome relatively uncharted. The past several years have seen an explosion in the development of generic and global approaches to study protein phosphorylation, thanks in large part to advances in robotics, mass spectrometry, and computational biology. As of July 2011, there were more than 60,000 nonredundant phosphorylation sites for human proteins annotated in a large repository. This explosion of data has, however, highlighted important issues regarding both the reliability of these types of identifications and the problem of assigning function to each of these phosphorylation events. Parallel advances in the identification of consensus sites for kinases, and systematic mapping of protein-protein interactions in signaling pathways, have provided complementary information that should help in obtaining a more holistic view of signaling. Here, we provide a perspective on system-wide approaches based on mass spectrometry to understand phosphoregulation.

Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells.

Protein kinase CK2 is a constitutively active protein serine/threonine kinase that is ubiquitously expressed and essential for the survival of eukaryotic cells. On the basis of its elevated expression in a number of human cancers and its ability to promote tumorigenesis in transgenic mice, CK2 has emerged as a promising candidate for molecular-targeted therapy. Accordingly, there has been considerable interest in identifying the cellular events that are regulated by CK2 and the cellular substrates of CK2 that are responsible for mediating its actions in cells. Large-scale phosphoproteomics studies are revealing extensive lists of candidate CK2 substrates on the basis that these proteins are phosphorylated at sites conforming to the consensus for phosphorylation by CK2. However, efforts to validate the vast majority of these candidates as bona fide physiological CK2 substrates have been hindered by the lack of systematic strategies to identify its direct substrates and manipulate its activity in intact cells. To overcome these limitations, we describe experimental procedures for isolating CK2 from bacteria and from mammalian cells to enable in vitro phosphorylation of candidate substrates. We also outline strategies for manipulating the levels and activity of CK2 in intact cells. Collectively, the methods that are presented in this chapter should enable the identification and characterization of CK2 substrates and CK2-regulated processes both in vitro and in living cells.

Evidence for regulation of mitotic progression through temporal phosphorylation and dephosphorylation of CK2alpha.

Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2alpha, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2alpha phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2alpha is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2alpha (CK2alpha-4D, CK2alpha-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2alpha (CK2alpha-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2alpha requires precise regulation to allow proper mitotic progression.