The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells.
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PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways. (+info)
Characterization of Chlamydomonas reinhardtii zygote-specific cDNAs that encode novel proteins containing ankyrin repeats and WW domains.
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Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles. (+info)
RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, an MHC class II immunodeficiency.
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The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions. (+info)
Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
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Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL. (+info)
Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor.
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Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-kappaB subunit p65 (RelA) and inhibits its transcriptional activity. RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait. We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay. RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain. RAI mRNA was preferentially expressed in human heart, placenta, and prostate. Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay. RAI inhibited the action of NF-kappaB p65 but not that of p53 in transient luciferase gene expression assays. Similarly, RAI inhibited the endogenous NF-kappaB activity induced by tumor necrosis factor-alpha. RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells. RAI protein appeared to be located in the nucleus and colocalized with NF-kappaB p65 that was activated by TNF-alpha. These observations indicate that RAI is another inhibitor of NF-kappaB in addition to IkappaB proteins and may confer an alternative mechanism of regulation. (+info)
INK4 cell cycle inhibitors direct transcriptional inactivation of NF-kappaB.
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The nuclear factor kappaB, a transcription factor regulating the expression of multiple genes including genes essential for cell cycle control, is found in most cells in a dormant state in the cytoplasm bound to the inhibitory family I kappaB via an ankyrin repeat domain. Stimulation of cells with a variety of inducers inactivates I kappaB proteins. The active dimeric NF-kappaB complex, often composed of 50- and 65-kilodalton subunits of the Rel family, translocates into the nucleus, where the NF-kappaBp65 subunit stimulates transcription. Here we report that a family of proteins containing ankyrin repeats, the inhibitors of Cdk4 (INK4) is able to bind NF-kappaBp65. The association of p16INK4 with NF-kappaBp65 is considerable in HeLa- or 293 cells, if the NF-kappaB inhibitor I kappaB alpha is degraded in response to TNFalpha stimulation. Overexpression of INK4 molecules suppresses the transactivational ability of NF-kappaB significantly. In contrast to INK4 proteins, the cell cycle inhibitor p27 enhances NF-kappaB transactivation activity. Thus, the effect of INK4 proteins on NF-kappaB function possibly modifies NF-kappaB mediated transcriptional activation of cell cycle associated factors. (+info)
The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.
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The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase. (+info)
Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.
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Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function. (+info)