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mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.

The vertebrate and neural-specific Ser/Arg (SR)-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells while weakening 3' splice site recognition and contributing to exon skipping in nonneural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis.

We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis.

SummaryThe pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, β-catenin, can physically associate as a ternary complex. This association stabilizes β-catenin via HDAC6 deacetylase activity, which leads to activation of β-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased β-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with GatewayTM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.

Association of serine/threonine phosphatases with regulatory proteins is a key component of their specificity, and the identification of these binding partners is critical to understanding phosphatases function and regulation. Affinity-purification/mass spectrometry (AP-MS) approaches have been and continue to be instrumental in identifying these interactors. Here, we review the general principles of AP-MS, and present two affinity-purification protocols compatible with subsequent mass spectrometry, namely FLAG purification, and the tandem affinity purification (TAP). We have successfully used these protocols for the identification of binding partners for PP2A, PP4 and PP6, and they should be amenable to the analysis of interactors for other phosphatases.

Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1–controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2- and ephrin-B1–expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling revealed that signaling between mixed EphB2- and ephrin-B1–expressing cells is asymmetric and that the distinct cell types use different tyrosine kinases and targets to process signals induced by cell-cell contact. We provide systems- and cell-specific network models of contact-initiated signaling between two distinct cell types.

Phosphorylation of histone H2AX on Ser 139 (gammaH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of gammaH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of gammaH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of gammaH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved gammaH2AX phosphatase.

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor α signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c·PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c·PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and γH2AX, but this activity is lower than that associated with the PP4c·PP4R2·PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2·PP4R3, and α4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

The CD133 antigen AC133/1 is a putative marker for enriching certain stem cells and cancer progenitor cells. Despite its wide application, the function and regulation of its expression remains unclear. Given the presence of its transcript in more differentiated cells, epigenetic regulation of CD133 transcripts alone cannot vouch for its utility as a marker. Therefore, understanding the regulation of CD133 protein expression is important for further defining the cellular state of tumor-initiating cells. We used the MAPLE system (Mak et al, Mol Cell Proteomics, 2010) to identify bona fide protein interaction partners of CD133. We present the microtubule-associated histone deacetylase 6 (HDAC6) as a physical interaction partner of CD133. This interaction was validated by co-immunoprecipitation/Western Blot analyses and by a membrane yeast two-hybrid (MYTH) assay. In order to understand the functional consequence of the HDAC6 and CD133 interaction, we used lentiviral short-hairpin RNAs to knockdown HDAC6 in multiple cell types, including the human epithelial colorectal adenocarcinoma lines Caco- 2 and HT-29 and in the retinoblastoma cell line Weri-Rb-1. Interestingly, we observed a significant reduction of CD133 protein expression. This reduction was shown to be dependent on HDAC6 deacetylase activity using a deacetlyase deficient HDAC6 mutant as well as when using the small molecule inhibitor of HDAC6, tubacin. HDAC6 promotes de-acetylation of alpha-tubulin to influence re-cycling of cell surface proteins. Furthermore, when we performed a CD133 MYTH screen against a cDNA library isolated from an adult human brain, we identified the late endosomal- and lysosomal-associated marker CD63 as an interaction partner of CD133. In fact, treatment of Caco-2 cells with tubacin resulted in trafficking of CD133 into endosomes for lysosomal degradation as determined by increased co-localization with CD63. In an attempt to study the affect of HDAC6 knockdown on cell viability, we noticed that CD133 knockdown significantly impacted cell proliferation and clonogenicity of Caco-2 cells. We determined that the cause of this is likely due to increased differentiation as determined by the level of alkaline phosphatase activity and by quantitative realtime PCR for markers of differentiated colon cells. To assess the tumorigenic potential of Caco-2 cells with reduced CD133 expression in vitro we performed soft agar colony-forming assay, which resulted in the loss of anchorage independent growth, a well-known hallmark of cancer. Together, these results suggest that CD133 can exercise a function in establishing and maintaining a primitive cancer cell state and loss or reduction of its expression marks more differentiated cancer cells that may be nontumorigenic. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A11.