A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners

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.

Download Full-text

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

Reproduction Fertility and Development ◽

10.1071/srb10abs326 ◽

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

Download Full-text

Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6633 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6633-6644 ◽

Cited By ~ 124

Author(s):

L Rui ◽

L S Mathews ◽

K Hotta ◽

T A Gustafson ◽

C Carter-Su

Keyword(s):

Growth Hormone ◽

Tyrosine Kinase ◽

Signaling Molecules ◽

Sh2 Domain ◽

Wild Type ◽

Yeast Two Hybrid ◽

Cos Cells ◽

Two Hybrid

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.

Download Full-text

The Use of the Yeast Two Hybrid System to Evaluate ErbB-3 Interactions with SH2 Domain Containing Proteins

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1998.9565 ◽

1998 ◽

Vol 251 (3) ◽

pp. 903-906 ◽

Cited By ~ 3

Author(s):

Joo-Yeon Yoo ◽

Anne W. Hamburger

Keyword(s):

Hybrid System ◽

Sh2 Domain ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

Download Full-text

dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX

Biochemical Journal ◽

10.1042/bj20060159 ◽

2006 ◽

Vol 398 (1) ◽

pp. 45-54 ◽

Cited By ~ 17

Author(s):

Ross Kinstrie ◽

Pamela A. Lochhead ◽

Gary Sibbet ◽

Nick Morrice ◽

Vaughn Cleghon

Keyword(s):

Chromatin Remodelling ◽

Drosophila Embryo ◽

Activation Loop ◽

Tyrosine Residue ◽

Yeast Two Hybrid ◽

Binding Partners ◽

C Terminus ◽

Dual Specificity ◽

Two Hybrid

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling.

Download Full-text

New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly

Journal of Cell Science ◽

10.1242/jcs.00425 ◽

2003 ◽

Vol 116 (11) ◽

pp. 2169-2178 ◽

Cited By ~ 40

Author(s):

S. Lu

Keyword(s):

Yeast Two Hybrid ◽

Binding Partners ◽

Two Hybrid ◽

Hybrid Screening

Download Full-text

Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03306-y ◽

2019 ◽

Vol 77 (13) ◽

pp. 2605-2620 ◽

Cited By ~ 1

Author(s):

Bethany Mason ◽

Susanne Flach ◽

Felipe R. Teixeira ◽

Raquel Manzano Garcia ◽

Oscar M. Rueda ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Genome Rearrangement ◽

Yeast Two Hybrid ◽

Binding Partners ◽

Functional Consequences ◽

Activity Loss ◽

F Box Protein ◽

Lrr Domain ◽

Two Hybrid

Abstract In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded by FBXL17 is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.

Download Full-text