CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response.

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Author Correction: Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.

Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment.

The human NDR family kinases control diverse aspects of cell growth, and are regulated through phosphorylation and association with scaffolds such as MOB1. Here, we report the crystal structure of the human NDR1 kinase domain in its non-phosphorylated state, revealing a fully resolved atypically long activation segment that blocks substrate binding and stabilizes a non-productive position of helix αC. Consistent with an auto-inhibitory function, mutations within the activation segment of NDR1 dramatically enhance in vitro kinase activity. Interestingly, NDR1 catalytic activity is further potentiated by MOB1 binding, suggesting that regulation through modulation of the activation segment and by MOB1 binding are mechanistically distinct. Lastly, deleting the auto-inhibitory activation segment of NDR1 causes a marked increase in the association with upstream Hippo pathway components and the Furry scaffold. These findings provide a point of departure for future efforts to explore the cellular functions and the mechanism of NDR1.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity.

The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser151 This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser151 in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.

Regulation of Protein Interactions by Mps One Binder (MOB1) Phosphorylation.

MOB1 is a multifunctional protein best characterized for its integrative role in regulating Hippo and NDR pathway signaling in metazoans and the Mitotic Exit Network in yeast. Human MOB1 binds both the upstream kinases MST1 and MST2 and the downstream AGC group kinases LATS1, LATS2, NDR1, and NDR2. Binding of MOB1 to MST1 and MST2 is mediated by its phosphopeptide-binding infrastructure, the specificity of which matches the phosphorylation consensus of MST1 and MST2. On the other hand, binding of MOB1 to the LATS and NDR kinases is mediated by a distinct interaction surface on MOB1. By assembling both upstream and downstream kinases into a single complex, MOB1 facilitates the activation of the latter by the former through a trans-phosphorylation event. Binding of MOB1 to its upstream partners also renders MOB1 a substrate, which serves to differentially regulate its two protein interaction activities (at least in vitro). Our previous interaction proteomics analysis revealed that beyond associating with MST1 (and MST2), MOB1A and MOB1B can associate in a phosphorylation-dependent manner with at least two other signaling complexes, one containing the Rho guanine exchange factors (DOCK6-8) and the other containing the serine/threonine phosphatase PP6. Whether these complexes are recruited through the same mode of interaction as MST1 and MST2 remains unknown. Here, through a comprehensive set of biochemical, biophysical, mutational and structural studies, we quantitatively assess how phosphorylation of MOB1A regulates its interaction with both MST kinases and LATS/NDR family kinases in vitro Using interaction proteomics, we validate the significance of our in vitro studies and also discover that the phosphorylation-dependent recruitment of PP6 phosphatase and Rho guanine exchange factor protein complexes differ in key respects from that elucidated for MST1 and MST2. Together our studies confirm and extend previous work to delineate the intricate regulatory steps in key signaling pathways.

MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway.

The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.

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.

BioID-based Identification of Skp Cullin F-box (SCF)ß-TrCP1/2 E3 Ligase Substrates.

The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(ß-TrCP1) and SCF(ß-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(ß-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(ß-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ß-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ß-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.

The PPFIA1-PP2A protein complex promotes trafficking of Kif7 to the ciliary tip and Hedgehog signaling.

The primary cilium is required for Hedgehog (Hh) signaling in vertebrates. Hh leads to ciliary accumulation and activation of the transmembrane protein Smoothened (Smo) and affects the localization of several pathway components, including the Gli family of transcriptional regulators, within different regions of primary cilia. Genetic analysis indicates that the kinesin protein Kif7 both promotes and inhibits mouse Hh signaling. Using mass spectrometry, we identified liprin-α1 (PPFIA1) and the protein phosphatase PP2A as Kif7-interacting proteins, and we showed that they were important for the trafficking of Kif7 and Gli proteins to the tips of cilia and for the transcriptional output of Hh signaling. Our results suggested that PPFIA1 functioned with PP2A to promote the dephosphorylation of Kif7, triggering Kif7 localization to the tips of primary cilia and promoting Gli transcriptional activity.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells.

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

Characterization of a modified ROCK2 protein that allows use of N6-ATP analogs for the identification of novel substrates.

BACKGROUND: The Rho-associated coiled-coil kinase-2 (ROCK2) is an important signaling transducer in the transmission of extracellular signals effecting organization of the actin cytoskeleton. ROCK2 has been implicated in numerous pathologies and the current focus is on understanding the molecular events that couple ROCK2 activity to biological function. To aid in the search for new ROCK2 substrates, we have developed an analog-sensitive (AS) ROCK2 protein that allows the use of selective ATP analogs that are not efficiently utilized by other protein kinases. RESULTS: The analog sensitive protein, M160A ROCK2, was highly active and could phosphorylate proteins from a cellular homogenate with γ32P-N6 (benzyl)ATP. We show the utility of this approach by identifying a putative ROCK2 substrate, elongation initiation factor-1-α1. We further show that the major site of ROCK2 phosphorylation of EIF1α1 is Thr432. CONCLUSIONS: Our work demonstrates that AS-ROCK2 could be useful in a systematic proteomic approach for identifying novel ROCK2 substrates.

Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

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/.

Control of the hippo pathway by Set7-dependent methylation of Yap.

Methylation of nonhistone proteins is emerging as a regulatory mechanism to control protein function. Set7 (Setd7) is a SET-domain-containing lysine methyltransferase that methylates and alters function of a variety of proteins in vitro, but the in vivo relevance has not been established. We found that Set7 is a modifier of the Hippo pathway. Mice that lack Set7 have a larger progenitor compartment in the intestine, coinciding with increased expression of Yes-associated protein (Yap) target genes. Mechanistically, monomethylation of lysine 494 of Yap is critical for cytoplasmic retention. These results identify a methylation-dependent checkpoint in the Hippo pathway.

The MYC-associated protein CDCA7 is phosphorylated by AKT to regulate MYC-dependent apoptosis and transformation.

Cell division control protein A7 (CDCA7) is a recently identified target of MYC-dependent transcriptional regulation. We have discovered that CDCA7 associates with MYC and that this association is modulated in a phosphorylation-dependent manner. The prosurvival kinase AKT phosphorylates CDCA7 at threonine 163, promoting binding to 14-3-3, dissociation from MYC, and sequestration to the cytoplasm. Upon serum withdrawal, induction of CDCA7 expression in the presence of MYC sensitized cells to apoptosis, whereas CDCA7 knockdown reduced MYC-dependent apoptosis. The transformation of fibroblasts by MYC was reduced by coexpression of CDCA7, while the non-MYC-interacting protein Δ(156-187)-CDCA7 largely inhibited MYC-induced transformation. These studies provide insight into a new mechanism by which AKT signaling to CDCA7 could alter MYC-dependent growth and transformation, contributing to tumorigenesis.

Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins.

Reversible phosphorylation events regulate critical aspects of cellular biology by affecting protein conformation, cellular localization, enzymatic activity and associations with interaction partners. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and other proteins, including scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. In this review we outline general considerations for successful AP-MS, and describe strategies that we have used to characterize the interactions of kinases and phosphatases in human cells.