Journal: Journal of Biological Chemistry

Article Title: Isoform Specificity of Ankyrin-B

doi: 10.1074/jbc.m506697200

Figure Lengend Snippet: FIGURE 1. Identification of an inter-domain interaction within ankyrin-B. A, domain organization of 220-kDa ankyrin-B. Ankyrin-B contains a membrane-binding domain, spectrin-binding domain, death domain, and C-terminal domain. The combination of death and C-terminal domain is termed the “regulatory domain.” B, ankyrin-B regulatory domain interacts with the membrane-binding domain in yeast two-hybrid assays. The ankyrin-B regulatory domain (fused with DNA-binding domain pAS2-1; Bait) was co- transformed into AH109 yeast strain with various ankyrin-B Prey constructs representing various ankyrin-B domains (fused to the DNA activation domain, pACT2). Positive inter- action was assessed by growth on media lacking adenine, histidine, leucine, tryptophan (-AHLT). We observed a positive interaction only when pAS2-1 regulatory domain was expressed with pACT2 membrane-binding domain.

Article Snippet: Fragments were PCR-amplified and inserted either in the pACT2 (GAL-4 activation domain vector) or pAS2-1 (GAL-4 DNA-binding domain vector).

Techniques: Membrane, Binding Assay, Transformation Assay, Construct, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Isoform Specificity of Ankyrin-B

doi: 10.1074/jbc.m506697200

Figure Lengend Snippet: FIGURE 3. Identification of the ankyrin-B membrane-binding domain site on the ankyrin-B regulatory domain. Yeast AH109 cells were co-transformed with ankyrin-B membrane-binding domain (fused with GAL-4 DNA activation domain) and one of ten Prey plasmids containing full-length or partial sequence of the ankyrin-B regulatory domain (amino acids 1445–1840). The death domain does not bind the membrane- binding domain while the C-terminal domain alone maintains binding affinity similar to the regulatory domain construct. The minimal binding region within the C-terminal domain is between amino acids 1556 and 1630. Deletion of this minimal binding region, D7, eliminates the intramolecular interaction. FIGURE 4. Identification of amino acids within the ankyrin-B C-terminal domain required for ankyrin-B inter-domain interaction. A, amino acid sequence within the C-terminal domain that contains membrane-binding domain activity. Amino acids cho- sen for alanine conversion are highlighted in red. Alanine-scanning mutants were gen- erated in the regulatory domain (pAS2-1) construct (see “Material and Methods” for details). B, a total of nine alanine-scanning mutants were screened for loss of binding to themembrane-bindingdomain(pACT2)revealingthataminoacidsGlu1597,Glu1598,and Asp1599 (regulatory 1597EEDAAA (pAS2-1)) are required for binding the membrane-bind- ing domain. The remaining eight mutants showed equivalent binding to the non-mu- tated regulatory domain.

Article Snippet: Fragments were PCR-amplified and inserted either in the pACT2 (GAL-4 activation domain vector) or pAS2-1 (GAL-4 DNA-binding domain vector).

Techniques: Membrane, Binding Assay, Transformation Assay, Activation Assay, Sequencing, Construct, Activity Assay

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: (A) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1ΔN3. Grey lines indicate RhoA-GTPase and DAD binding regions. G-GTPase binding domain, DID-Diaphanous inhibitory domain, FH1, FH2, FH3-Formin Homology domains, DAD-Diaphanous Auto-inhibitory Domain. Start positions of the domains are depicted. (B) Phb2 identified as mDia1 interacting protein in a yeast two hybrid screen. PJ69-4A was co-transformed with Phb2-AD and mDia1ΔN3-BD (positive GAL4 reconstitution) or empty-BD (negative GAL4 reconstitution) and four colonies per reconstitution were screened for ADE2 and LacZ reporters on -Trp/-Leu/-Ade and -Trp/-Leu+X-Gal plates respectively. Positive control “P”- Drosophila Batman-AD and GAGA factor-BD, negative control “N”-empty-AD and empty BD. Induction of ADE2 is indicated by growth and induction of LacZ is indicated by blue colour. Trp-Tryptophan, Leu-Leucine, Ade-Adenine. AD-Activation domain, BD binding domain. (C) Domain structure of Phb2-FL and Phb2-Y2H. HYD-Hydrophobic region, PHB-Prohibitin domain, CC-Coiled coil domain. (D) Co-IP of exogenous Phb2 and mDia1ΔN3 to confirm the interaction. HEK293T, co-transfected with mDia1ΔN3 and Phb2-Y2H, and pulled down with anti-Flag antibody. (E,F) Reciprocal IP of endogenous mDia1 and Phb2 to identify stage-specific interaction. Lysates from proliferating MB (GM), MT in differentiation medium for 24 (D24), 36, (D36), 72 (D72) and 120 (D120) hours were harvested and subjected to IP with anti-mDia1 (E) or ant-Phb2 (F) antibodies. (G) LC-MS/MS analysis of mDia1-interacting proteins in myoblasts (MB) and myotubes (MT). Venn diagram represents the number of proteins that bind mDia1 in MB or MT or both MB and MT. (H) Phb2 peptides identified in MT lysates by LC-MS/MS analysis of mDia1 IP proteins. Peptides identified in the first biological replicate are indicated in red, peptides identified in the second and third replicate are shown in blue and those peptides common in all three replicates are underlined in Phb2 full-length aa sequence (NCBI Reference Sequence # NP_031557.2). The numbers represent aa position on mDia1 or Phb2 (A, C).

Article Snippet: Saccharomyces cerevisiae strain PJ69-4A was co transformed with mDia1ΔN3-BD (pGBKT7–GAL4 Binding domain vector) and Matchmaker 7day old mouse embryonic cDNA library cloned in pACT2 (AD-GAL4 Activation domain vector) (Clonetech) as per manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay, Two Hybrid Screening, Transformation Assay, Positive Control, Negative Control, Activation Assay, Co-Immunoprecipitation Assay, Transfection, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation

doi: 10.1101/283044

Figure Lengend Snippet: Yeast two-hybrid screen to identify novel mDia1-interacting proteins. A GAL4 hybrid reconstitution assay with putative mDia1 interactors was performed to study the induction of reporters ADE2 and LacZ on -Trp/-Leu/-Ade and -Trp/-Leu+X-Gal respectively. PJ69-4A was co transformed with interacting protein plasmid and mDia1ΔN3-BD (positive reconstitution) or empty BD vector (negative reconstitution). Four colonies per per reconstitution assay (positive and negative) were screened on selection plates. PJ69-4A co-transformed with bona fide interacting proteins Drosophila Batman-AD and GAGA factor-BD served as a positive control “P” for reporter expression and PJ69-4A co-transformed with empty pACT2 and pGBKT7 vectors served as a negative control “N”. Induction of ADE2 reporter is indicated by growth and induction of LacZ expression is indicated by blue colour in the colonies on the selection plates. Seven library clones that induced the reporter expression were finally selected from the screen as mDia1-interacting proteins of interest. (A) Profilin1 (Pfn1), known interactor for mDia1 (B) Cadherin11 (Cdh11) (C) Niemann Pick Type C2 (Npc2) (D) Leukocyte receptor cluster (LRC) member 8 (Leng8) (E) Growth factor receptor bound protein 2 (Grb2) (F) Protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (p58 repressor) (Prkrir) (G) Cytochrome c1 (Cyc1) were identified as mDia1-interacting proteins. Trp-Tryptophan, Leu-Leucine, Ade-Adenine.

Article Snippet: Saccharomyces cerevisiae strain PJ69-4A was co transformed with mDia1ΔN3-BD (pGBKT7–GAL4 Binding domain vector) and Matchmaker 7day old mouse embryonic cDNA library cloned in pACT2 (AD-GAL4 Activation domain vector) (Clonetech) as per manufacturer’s instructions.

Techniques: Two Hybrid Screening, Reconstitution Assay, Transformation Assay, Plasmid Preparation, Selection, Positive Control, Expressing, Negative Control, Clone Assay