Visualizing protein partnerships in living cells and organisms

Lowder, Melissa A.; Appelbaum, Jacob; Hobert, Elissa M.; Schepartz, Alanna

doi:10.1016/j.cbpa.2011.10.024

Cited by 33 publications

(32 citation statements)

References 47 publications

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“…Such screens include, but are not limited to, split molecule complementation (e.g., split GFP; ref. 33), chemical and photo crosslinking followed by specific immunoprecipitation to lock transient interactions in place (34), and the fusion of a bait protein with molecules that can modify and "mark" neighboring proteins (e.g., BirA; ref. 35).…”

Section: Discussionmentioning

confidence: 99%

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

Delker¹,

Zhou²,

Strikoudis³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.ubiquitin | immunology | lymphocyte M ost large-scale protein-protein interaction data published to date has been produced with yeast two-hybrid assays (1-3) and in vivo pull-down approaches followed by mass spectrometry to identify proteins that coprecipitate with the protein of interest. Although these purification methods were first developed for small-scale protein identification experiments, they have been successfully adapted for use in genome-wide proteomics studies (4-6). Proteins that are poorly soluble or insoluble when ectopically expressed are least amenable to characterization using these tools. Improper folding or exposure of hydrophobic domains can result in insolubility when proteins are expressed individually; however, soluble complexes can form upon coexpression of a native partner. Based on this idea, we have devised a high-throughput protein-protein interaction screen, which involves the coexpression and cofolding of two proteins: an unknown protein expressed from a cDNA library and a known, insoluble "bait" fused to a protein that confers antibiotic resistance. Thus, solubility is directly linked to a traceable marker. We apply this technique to identify cofactors for activation-induced cytidine deaminase (AID), an enzyme that deaminates deoxycytidines in single-stranded DNA.AID initiates both somatic hypermutation (SHM) and classswitch recombination (CSR) during antibody diversification (7,8). A number of cofactors have been identified by a combination of genetic and proteomic approaches, which suggest that AID synchronizes with other broadly important cellular pathways including, but not limited to, RNA splicing and processing (9, 10), cellular trafficking (11)(12)(13)(14), and transcription/RNA polymerase stalling (8,(15)(16)(17); however, a concise picture of the players involved in the AID reaction and the...

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Section: Discussionmentioning

confidence: 99%

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

Delker¹,

Zhou²,

Strikoudis³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…To gain further insight into the proximity of these associations, we used a proximity ligation assay that detects native protein-protein associations within 40 nm. 25 The strength of the output signal in PLA can vary as a function of antibody affinities, molecular orientations, and DNA amplification, but specificity is high. ADAP-talin associations produced a clear, specific PLA signal, with many bright red fluorescent dots indicating close proximity ( Figure 2C).…”

Section: Org Frommentioning

confidence: 99%

“…Platelet proximity ligation assays (PLA) that report on endogenous proteinprotein associations at ,40 nm 25 were performed by following the manufacturer's recommendations (O-Link Bioscience, Uppsala, Sweden) using oligonucleotide-conjugated secondary antibodies against mouse and rabbit primary antibodies. Protein-protein associations were detected by microscopy as bright red dots.…”

Section: Microscopic Image Acquisition and Analysesmentioning

confidence: 99%

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

Kasirer-Friede

Kang

Kahner

et al. 2014

Blood

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Key Points• ADAP interacts with talin and kindlin-3 in platelets.• ADAP is a hematopoietic component of the molecular machinery that promotes activation of and stable fibrinogen binding to aIIbb3.ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase-associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin aIIbb3 activation in platelets, which lack SKAP1, suggesting an ADAP integrinregulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-b cytoplasmic tail-binding proteins involved in aIIbb3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with aIIbb3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing aIIbb3, talin, PAR1, and kindlin-3, it associated with an aIIbb3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to aIIbb3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet aIIbb3 through interactions with talin and kindlin-3. (Blood. 2014;123(20): 3156-3165) IntroductionIntegrins engage in bidirectional signaling. Fibrinogen binding to integrins is regulated by inside-out signals initiated by agonist receptors, a process often called "integrin activation." In turn, ligandbound integrins transduce outside-in signals to regulate cellular responses, among them cytoskeletal reorganization. 1 In platelets, integrin aIIbb3 bidirectional signaling is required for efficient hemostatic platelet responses to vascular injury. Full inside-out signaling can promote increases in integrin affinity through conformational changes and increases in integrin avidity through receptor clustering; the relative contribution of each process can vary with the integrin and the cell studied.1,2 Recently, talin and kindlin, 2 adapter proteins that bind to integrin-b cytoplasmic tails, have emerged as essential regulators of integrin activation. Talin is a 280 kDa protein of the FERMT4 (4.1/ ezrin/radixin, moesin domain T4) family composed of head (amino acids 1-433) and rod (482-2541) domains that are normally clasped but upon relief of auto-inhibition can bind via the Ferm3 phospho tyrosinebinding subdomain to regions of integrin b...

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“…Protein-protein interactions were detected by using a Duolink PLA kit [Olink Bioscience, Uppsala, Sweden; Lowder et al, 2011), according to the manufacturer's protocol. Briefly, cells were grown, fixed, and permeabilized, as described in the immunofluorescence microscopy section.…”

Section: Proximity Ligation Assaymentioning

confidence: 99%

Mitotic Golgi translocation of ERK1c is mediated by PI4KIIIβ/14-3-3γ shuttling complex

Wortzel

Hanoch

Porat

et al. 2015

Journal of Cell Science

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Golgi fragmentation is a highly regulated process that allows division of the Golgi complex between the two daughter cells. The mitotic reorganization of the Golgi is accompanied by a temporary block in Golgi functioning, as protein transport in and out of the Golgi stops. Our group has previously demonstrated the involvement of the alternatively spliced variants ERK1c and MEK1b (ERK1 is also known as MAPK3, and MEK1 as MAP2K1) in mitotic Golgi fragmentation. We had also found that ERK1c translocates to the Golgi at the G2 to M phase transition, but the molecular mechanism underlying this recruitment remains unknown. In this study, we narrowed the translocation timing to prophase and prometaphase, and elucidated its molecular mechanism. We found that CDK1 phosphorylates Ser343 of ERK1c, thereby allowing the binding of phosphorylated ERK1c to a complex that consists of PI4KIIIβ (also known as PI4KB) and the 14-3-3γ dimer (encoded by YWHAB). The stability of the complex is regulated by protein kinase D (PKD)-mediated phosphorylation of PI4KIIIβ. The complex assembly induces the Golgi shuttling of ERK1c, where it is activated by MEK1b, and induces Golgi fragmentation. Our work shows that protein shuttling to the Golgi is not completely abolished at the G2 to M phase transition, thus integrating several independent Golgiregulating processes into one coherent pathway.

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Visualizing protein partnerships in living cells and organisms

Cited by 33 publications

References 47 publications

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

Mitotic Golgi translocation of ERK1c is mediated by PI4KIIIβ/14-3-3γ shuttling complex

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