Identification of the multivalent PDZ domain protein PDZK1 as a binding partner of sodium‐coupled monocarboxylate cotransporter 2 (SMCT2) by yeast two‐hybrid assay

A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.

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Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

International Journal of Molecular Sciences ◽

10.3390/ijms21093193 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3193 ◽

Cited By ~ 1

Author(s):

Juan Luis Araujo-Garrido ◽

Fernando Baisón-Olmo ◽

Joaquín Bernal-Bayard ◽

Francisco Romero ◽

Francisco Ramos-Morales

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Salmonella Enterica Serovar Typhimurium ◽

Yeast Two Hybrid ◽

Microtubule Network ◽

Binding Partner ◽

Tubulin Binding ◽

Serovar Typhimurium ◽

Novel Target ◽

Two Hybrid

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

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The Multivalent PDZ Domain-containing Protein PDZK1 Regulates Transport Activity of Renal Urate-Anion Exchanger URAT1 via Its C Terminus

Journal of Biological Chemistry ◽

10.1074/jbc.m406724200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45942-45950 ◽

Cited By ~ 127

Author(s):

Naohiko Anzai ◽

Hiroki Miyazaki ◽

Rie Noshiro ◽

Suparat Khamdang ◽

Arthit Chairoungdua ◽

...

Keyword(s):

Anion Exchanger ◽

Apical Membrane ◽

Organic Anion ◽

Pdz Domain ◽

Hek293 Cells ◽

Transport Activity ◽

Yeast Two Hybrid ◽

Urate Transport ◽

Anion Transporter ◽

Two Hybrid

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002)Nature417, 447–452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95,Drosophiladiscs-large protein,Zonula occludensprotein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay,in vitrobinding assay and surface plasmon resonance analysis (KD= 1.97–514 nm). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in theVmaxof urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.

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The Four and a Half LIM Domain Protein 2 (FHL2) Interacts with CALM and Is Highly Expressed in AML with Complex Aberrant Karyotpes.

Blood ◽

10.1182/blood.v108.11.4337.4337 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4337-4337 ◽

Cited By ~ 1

Author(s):

Zlatana Pasalic ◽

Belay Tizazu ◽

Leticia Archangelo ◽

Alexandre Krause ◽

Greif Philipp ◽

...

Keyword(s):

Transcription Factor ◽

Fusion Protein ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Luciferase Reporter ◽

Lim Domain ◽

Yeast Two Hybrid ◽

Lim Domain Protein ◽

Domain Protein ◽

Two Hybrid

Abstract The balanced chromosomal translocation t(10;11)(p13;q14) results in the CALM/AF10 fusion gene. This translocation is found in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL) and malignant lymphoma. The CALM/AF10 fusion gene has recently been shown to cause an aggressive biphenotypic leukemia in a murine bone marrow transplant model. The CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) gene product is a clathrin assembly protein which plays a role in clathrin mediated endocytosis and trans Golgi network trafficking. AF10 is a putative transcription factor most likely involved in processes related to chromatin organization and has polycomb group gene like properties. To learn more about the function of the CALM/AF10 fusion protein, we searched for protein interaction partners of CALM. In a yeast two hybrid screen the four and a half LIM domain protein FHL2 was identified as putative CALM interacting partner. The CALM FHL2 interaction was confirmed by co-transformation assay in yeast and by GST-pulldown experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294 to 335 of CALM using the yeast two hybrid assay. In co-localization studies with transiently expressed fluorescent protein tagged CALM and FHL2, both proteins showed cytoplasmatic localization. Expression analysis (Affymetrix based) in different AML subtypes showed a significantly higher expression of FHL2 in AML with complex aberrant karyotypes compared to AML with normal karyotypes or balanced chromosomal translocations like the t(8;21), inv(16) or t(15;17). FHL2, which is also known as DRAL (downregulated in rhabdomyosarcoma LIM protein), is a TP53 responsive gene known to interact with numerous proteins in both the nucleus and the cytoplasm and can function as a transcriptional cofactor. Known FHL2 interactors include TP53, BRCA1, PLZF (promyelocytic leukemia zinc finger protein), the proto-oncogene SKI1 and beta-catenin. High expression of FHL2 in breast cancer has recently been shown to be associated with an adverse prognosis. CALM has been shown to shuttle between the nucleus and the cytoplasm because inhibition of CREM-mediated nuclear export by leptomycin B leads to the accumulation of CALM in the nucleus. Reporter gene assays using a GAL4-DNA binding domain CALM fusion protein and a GAL4 responsive luciferase reporter were able to demonstrate a transcriptional activation function of CALM. We are currently investigation the effect of FHL2 co-expression on this aspect of the CALM function. It is thus conceivable that FHL2 is playing an important role in CALM/AF10-mediated leukemogenesis by tethering the CALM/AF10 fusion protein to various nuclear transcription factor complexes.

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Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.

Blood ◽

10.1182/blood.v116.21.1444.1444 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1444-1444

Author(s):

Denisa Urban ◽

Ling Li ◽

James Wasmuth ◽

Hilary Christensen ◽

John Parkinson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Human Platelets ◽

Multiprotein Complex ◽

Yeast Two Hybrid ◽

Granule Formation ◽

Reading Frame ◽

Binding Partner ◽

Library Screen ◽

Two Hybrid ◽

Granule Release

Abstract Abstract 1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis. Disclosures: No relevant conflicts of interest to declare.

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Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)

Biochemical Journal ◽

10.1042/bj3610443 ◽

2002 ◽

Vol 361 (3) ◽

pp. 443-450 ◽

Cited By ~ 45

Author(s):

Alesia M. HRUSKA-HAGEMAN ◽

John A. WEMMIE ◽

Margaret P. PRICE ◽

Michael J. WELSH

Keyword(s):

Ion Channel ◽

Mammalian Cells ◽

Hippocampal Neurons ◽

Sequence Similarity ◽

Expression System ◽

Pdz Domain ◽

Mammalian Brain ◽

Na Channel ◽

Yeast Two Hybrid ◽

Two Hybrid

Neuronal members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels include the mammalian brain Na+ channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related αENaC or βENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

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Acheron, an novel LA antigen family member, binds to cask and forms a complex with id transcription factors

Cellular & Molecular Biology Letters ◽

10.2478/s11658-008-0046-1 ◽

2009 ◽

Vol 14 (2) ◽

Cited By ~ 12

Author(s):

Haifeng Weng ◽

Chul Kim ◽

Christos Valavanis ◽

Zhaohui Wang ◽

Lawrence Schwartz

Keyword(s):

Transcription Factors ◽

Family Member ◽

Cdna Library ◽

Mouse Embryo ◽

Yeast Two Hybrid ◽

Binding Partner ◽

C Terminus ◽

La Antigen ◽

Two Hybrid

AbstractAcheron, a Lupus antigen ortholog, was identified as a novel death-associated transcript from the intersegmental muscles of the mothManduca sexta. Acheron is phylogenetically-conserved and represents a new sub-family of Lupus antigen proteins. Acheron is expressed predominantly in neurons and muscle in vertebrates, and regulates several developmental events including myogenesis, neurogenesis and possibly metastasis. Using Acheron as bait, we performed a yeast two-hybrid screen with a mouse embryo cDNA library and identified CASK-C, a novel CASK/Lin-2 isoform, as an Acheron binding partner. Acheron and CASK-C bind via the C-terminus of Acheron and the CaMKII-like domain of CASK-C. Co-immunoprecipitation assays verify this interaction and demonstrate that Acheron also forms a complex with all members of the Id (inhibitor of differentiation) proteins. Taken together, these data suggest a mechanism by which Acheron may regulate development and pathology.

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The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis

Blood ◽

10.1182/blood-2012-05-431205 ◽

2012 ◽

Vol 120 (25) ◽

pp. 5032-5040 ◽

Cited By ~ 59

Author(s):

Denisa Urban ◽

Ling Li ◽

Hilary Christensen ◽

Fred G. Pluthero ◽

Shao Zun Chen ◽

...

Keyword(s):

Immunofluorescence Microscopy ◽

Binding Protein ◽

Yeast Two Hybrid ◽

Binding Partner ◽

Late Endosomes ◽

Arc Syndrome ◽

Membrane Bound ◽

Golgi Network ◽

Two Hybrid

Abstract Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

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Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function

eLife ◽

10.7554/elife.14222 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 18

Author(s):

Bo Zhao ◽

Zizhen Wu ◽

Ulrich Müller

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Cell Function ◽

Yeast Two Hybrid ◽

Cochlear Hair Cells ◽

Unknown Mechanism ◽

Binding Partner ◽

Stochastic Optical Reconstruction Microscopy ◽

Optical Reconstruction ◽

Two Hybrid

Cochlear hair cells convert sound-induced vibration into electrical signals. FAM65B mutations cause hearing loss by an unknown mechanism. Using biochemistry and stochastic optical reconstruction microscopy (STORM), we show here that Fam65b oligomers form a circumferential ring near the basal taper of the mechanically sensitive stereocilia of murine hair cells. Taperin, a second protein near the taper, forms a dense-core-like structure that is disrupted in the absence of Fam65b. Stereocilia of Fam65b-deficient murine hair cells start to develop, but mechanotransduction is affected and stereocilia deteriorate. Yeast-two-hybrid screens identify RhoC as a Fam65b binding partner. RhoC co-localizes with Fam65b in stereocilia and regulates Fam65b oligomerization. Binding to RhoC and oligomerization are critical for Fam65b function. Our findings thus reveal a highly organized compartment near the base of stereocilia that is critical for hair cell function and affected in disease.

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