326. SPIF - A NOVEL TESTIS-SPECIFIC GENE AND ITS INTERACTION WITH PKA

2010 ◽  
Vol 22 (9) ◽  
pp. 126
Author(s):  
S. J. Tannock ◽  
E. A. McLaughlin ◽  
R. J. Aitken ◽  
S. D. Roman
Keyword(s):  
Northern Blotting ◽  
Full Length ◽  
Specific Gene ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Truncated Form ◽  
Binding Partners ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Two Hybrid

The activation of protein kinase A (PKA) is strongly implicated in capacitation and sperm motility. However, the full pathway is yet to be elucidated. To identify potential PKA binding partners in sperm, a yeast two-hybrid assay was performed using the testis specific catalytic subunit (Cs) of PKA as the ‘bait’ to screen a mouse testis cDNA library. A novel cDNA clone termed Sperm PKA Interacting Factor (SPIF) was identified from the screen on three separate occasions. The interaction was confirmed by a protein pull-down using a C-terminal recombinant protein to SPIF and a PKACs antibody. During cloning and sequence analysis, SPIF was found to contain two isoforms; a full length (4770 bp) and a truncated form (2784 bp) with alternate start sites and an identical 3′ end, with only the full length isoform containing the PKA binding motif. SPIF was found to be testis specific using PCR and Northern Blotting with high expression levels in round spermatids and adult testis. The interaction between SPIF and PKA was further demonstrated with protein co-localisation in round spermatids and in the midpiece and flagellum of mouse sperm. In summary, we have identified a novel testis specific gene that in concert with PKA could prove to be an essential link in the incomplete capacitation pathway

A novel testis protein, RSB-66, interacting with INCA1 (inhibitor of Cdk interacting with cyclin A1)

2008 ◽  
Vol 86 (4) ◽  
pp. 345-351 ◽  
Author(s):  
Xu Chen ◽  
Tinghui Hu ◽  
Gang Liang ◽  
Maojun Yang ◽  
Shudong Zong ◽  
...  
Keyword(s):  
Round Spermatid ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
Cyclin A1 ◽  
Yeast Two Hybrid System ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Α Helix ◽  
Two Hybrid

rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The α helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.

scholarly journals Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses

2000 ◽  
Vol 149 (7) ◽  
pp. 1419-1432 ◽  
Author(s):  
Ute Schaeper ◽  
Niels H. Gehring ◽  
Klaus P. Fuchs ◽  
Martin Sachs ◽  
Bettina Kempkes ◽  
...  
Keyword(s):  
Binding Site ◽  
Mdck Cells ◽  
Mapk Pathway ◽  
Biological Response ◽  
Adaptor Protein ◽  
Branching Morphogenesis ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Interaction Sites ◽  
Two Hybrid

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met–specific branching morphogenesis. It associates directly with c-Met via the c-Met–binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met–binding site is localized to a 13–amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met–binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1–specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

A Method to Generate Full-Length cDNA Molecules from Yeast Two-Hybrid Clones and RACE Products

1999 ◽  
Vol 268 (1) ◽  
pp. 161-162 ◽  
Author(s):  
Charles S. Hemenway ◽  
Benjamin W. Halligan ◽  
Grahame C.D. Gould
Keyword(s):  
Full Length ◽  
Yeast Two Hybrid ◽  
Full Length Cdna ◽  
Hybrid Clones ◽  
Two Hybrid

scholarly journals Ebolavirus VP35 Interacts with the Cytoplasmic Dynein Light Chain 8

2009 ◽  
Vol 83 (13) ◽  
pp. 6952-6956 ◽  
Author(s):  
Toru Kubota ◽  
Mayumi Matsuoka ◽  
Tsung-Hsien Chang ◽  
Mike Bray ◽  
Steven Jones ◽  
...  
Keyword(s):  
Life Cycle ◽  
Light Chain ◽  
Viral Protein ◽  
Cytoplasmic Dynein ◽  
Type I ◽  
Dynein Light Chain ◽  
Yeast Two Hybrid ◽  
Binding Partners ◽  
Mapping Analysis ◽  
Two Hybrid

ABSTRACT The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains

1996 ◽  
Vol 109 (10) ◽  
pp. 2493-2498 ◽  
Author(s):  
D.A. Carpenter ◽  
W. Ip
Keyword(s):  
Intermediate Filaments ◽  
Hybrid System ◽  
Weak Interaction ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Building Blocks ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons. Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin. A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L. Interactions among the full length NF triplet proteins exhibited more specificity. Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain. No homologous or heterologous dimerization of NF-M and NF-H was detectable. These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks. They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains.

scholarly journals Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor

2003 ◽  
Vol 372 (2) ◽  
pp. 453-463 ◽  
Author(s):  
Marianne TARDIF ◽  
Laurence BROUCHON ◽  
Marie-Josèphe RABIET ◽  
François BOULAY
Keyword(s):  
Active Form ◽  
Hybrid Approach ◽  
Full Length ◽  
Actin Dynamics ◽  
Yeast Two Hybrid ◽  
C5a Receptor ◽  
Wiskott Aldrich Syndrome ◽  
C5a Anaphylatoxin ◽  
Syndrome Protein ◽  
Two Hybrid

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott–Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1–321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active ‘open conformation’, the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

scholarly journals A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas

2007 ◽  
Vol 176 (6) ◽  
pp. 819-829 ◽  
Author(s):  
Lai-Wa Tam ◽  
Nedra F. Wilson ◽  
Paul A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Cell Fractionation ◽  
Cyclin Dependent Kinase ◽  
Yeast Two Hybrid ◽  
Flagellar Assembly ◽  
Two Hybrid

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.

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