scholarly journals Binding of Integrin α6β4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

1999 ◽  
Vol 147 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Dirk Geerts ◽  
Lionel Fontao ◽  
Mirjam G. Nievers ◽  
Roel Q.J. Schaapveld ◽  
Patricia E. Purkis ◽  
...  
Keyword(s):  
Binding Site ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Cytoplasmic Domain ◽  
Actin Binding ◽  
Yeast Two Hybrid ◽  
Dot Blot ◽  
Intermediate Filament Proteins ◽  
Two Hybrid

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the α6β4 integrin and have shown that the cytoplasmic domain of the β4 subunit associates with an NH2-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with α6β4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that β4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the β4 cytoplasmic domain. Mapping of the β4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that β4 can compete out the plectin ABD fragment from its association with F-actin. The ability of β4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when β4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

2008 ◽  
Vol 183 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Noveera T. Ahmed ◽  
Chunlei Gao ◽  
Ben F. Lucker ◽  
Douglas G. Cole ◽  
David R. Mitchell
Keyword(s):  
Binding Site ◽  
Intraflagellar Transport ◽  
Site Formation ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
B Subunit ◽  
Dynein Arms ◽  
Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

scholarly journals A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

2006 ◽  
Vol 17 (4) ◽  
pp. 1971-1984 ◽  
Author(s):  
Michael G. Clark ◽  
Joseph Teply ◽  
Brian K. Haarer ◽  
Susan C. Viggiano ◽  
David Sept ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Interacting Protein ◽  
Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

scholarly journals Role of Binding of Plectin to the Integrin β4 Subunit in the Assembly of Hemidesmosomes

2004 ◽  
Vol 15 (3) ◽  
pp. 1211-1223 ◽  
Author(s):  
J. Koster ◽  
S. van Wilpe ◽  
I. Kuikman ◽  
S.H.M. Litjens ◽  
A. Sonnenberg
Keyword(s):  
Binding Sites ◽  
Cytoplasmic Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Integrin Β4 ◽  
Fibronectin Type Iii ◽  
Intramolecular Association ◽  
Two Hybrid ◽  
Fibronectin Type

We have previously shown that plectin is recruited into hemidesmosomes through association of its actin-binding domain (ABD) with the first pair of fibronectin type III (FNIII) repeats and a small part of the connecting segment (residues 1328–1355) of the integrin β4 subunit. Here, we show that two proline residues (P1330 and P1333) in this region of the connecting segment are critical for supporting β4-mediated recruitment of plectin. Additional binding sites for the plakin domain of plectin on β4 were identified in biochemical and yeast two-hybrid assays. These sites are located at the end of the connecting segment (residues 1383–1436) and in the region containing the fourth FNIII repeat and the C-tail (residues 1570–1752). However, in cells, these additional binding sites cannot induce the assembly of hemidesmosomes without the interaction of the plectin-ABD with β4. Because the additional plectin binding sites overlap with sequences that mediate an intramolecular association of the β4 cytoplasmic domain, we propose that they are not accessible for binding and need to become exposed as the result of the binding of the plectin-ABD to β4. Furthermore, these additional binding sites might be necessary to position the β4 cytoplasmic domain for an optimal interaction with other hemidesmosomal components, thereby increasing the efficiency of hemidesmosome assembly.

scholarly journals Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

1999 ◽  
Vol 341 (2) ◽  
pp. 257-263 ◽  
Author(s):  
Mark D. BASS ◽  
Beverley J. SMITH ◽  
Sally A. PRIGENT ◽  
David R. CRITCHLEY
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Leucine Zipper ◽  
Peptide Sequence ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Linear Peptide ◽  
Sds Page ◽  
Α Helix ◽  
Two Hybrid

Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic α-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites. To test the possibility that this might constitute a leucine zipper involved in vinculin binding, we mutated each leucine residue to an alanine. The results showed that this leucine repeat is not essential to the interaction between talin and vinculin. We also used the yeast two-hybrid system to define further the talin-binding site within vinculin residues 1-258. C-terminal deletions made in accordance with exon boundaries showed that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin. However, all N-terminal deletions abolished binding. The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain.

scholarly journals Functional Domains of the Drosophila Bicaudal-D Protein

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 713-724
Author(s):  
Junyoung Oh ◽  
Katalin Baksa ◽  
Ruth Steward
Keyword(s):  
Intermediate Filament ◽  
Protein Complex ◽  
Ovarian Function ◽  
Function Analysis ◽  
Heptad Repeat ◽  
Functional Importance ◽  
Yeast Two Hybrid ◽  
Intermediate Filament Proteins ◽  
Multimeric Protein ◽  
Two Hybrid

Abstract The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity. Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte. The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains. We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-DH) deleted for each of the heptad repeat domains in a Bic-D null background. Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in evolution. The analysis of females heterozygous for Bic-DH and the existing alleles Bic-DPA66 or Bic-DR26 reveals that Bic-DR26 as well as some of Bic-DH transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers.

scholarly journals Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

1991 ◽  
Vol 114 (3) ◽  
pp. 481-491 ◽  
Author(s):  
F M Pavalko ◽  
K Burridge
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Site ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Stress Fibers ◽  
Actin Binding ◽  
Cell Periphery

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.

scholarly journals Further characterization of the interaction between the cytoskeletal proteins talin and vinculin

2002 ◽  
Vol 362 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Mark D. BASS ◽  
Bipin PATEL ◽  
Igor G. BARSUKOV ◽  
Ian J. FILLINGHAM ◽  
Robert MASON ◽  
...  
Keyword(s):  
Binding Site ◽  
Intramolecular Interaction ◽  
Binding Activity ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Yeast Two Hybrid ◽  
Temperature Dependent ◽  
Two Hybrid ◽  
Actin Binding Site

The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1—VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498–636; VBS2, residues 727–965; and VBS3, residues 1943–2157) each bind tightly to the same or overlapping sites within vinculin1–258. A short synthetic talin VBS3 peptide (residues 1944–1969) was sufficient to inhibit binding of a 125I-labelled talin VBS3 polypeptide to vinculin1–258, and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin1–258. Binding of the 125I-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by 1M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin1–258 using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1–131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh—Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh—Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

Sign in / Sign up

Export Citation Format

Share Document