The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells.
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)
Cloning and characterization of androgen receptor coactivator, ARA55, in human prostate.
Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors. We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system. ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-beta1-inducible gene. Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a ligand-dependent manner. Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nM dihydrotestosterone or its antagonists such as 100 nM 17beta-estradiol or 1 microM hydroxyflutamide. Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR. Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells. Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation. (+info)
The LIM protein, CRP1, is a smooth muscle marker.
LIM domains are double zinc-finger motifs found in many proteins that play central roles in cell differentiation. Members of the cysteine-rich protein (CRP) family display two LIM domains and are implicated in muscle development. Here we describe the characterization of one member of this family, CRP1, in the mouse. We have isolated and sequenced murine cDNAs that encode CRP1. We have determined by Northern analysis and in situ hybridization that CRP1 expression is developmentally regulated in the embryonic mouse and displays organ specific regulation in adults. The gene encoding CRP1 is expressed in the smooth muscle cells (SMCs) of the dorsal aorta at E9.5, thus illustrating that CRP1 is an early marker for SMC differentiation at that site. As development proceeds, CRP1 transcripts are observed throughout the SMC lineage, with minimal, transient expression detected in skeletal and cardiac muscle. Interestingly, although several markers of mature smooth muscle are already expressed, CRP1 expression in the bladder is not upregulated until the onset of bladder expansion at embryonic day 16.5, at which time its expression becomes very prominent. CRP1 expression persists into adulthood with prominent expression observed in both vascular and visceral smooth muscle. The results reported here define CRP1 as a general marker of smooth muscle lineages. (+info)
Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain.
The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases. (+info)
Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma.
Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers. (+info)
Muscle LIM protein: expressed in slow muscle and induced in fast muscle by enhanced contractile activity.
To identify early changes in gene expression during the fast-to-slow transition induced by chronic low-frequency stimulation, total RNA was extracted from 12-h-stimulated tibialis anterior (TA) muscles of rats and amplified by differential display RT-PCR. Among the signals of differentially expressed mRNAs, a cDNA approximately 300 bp in length, which was almost undetectable in control TA muscles but prominent in stimulated TA and normal soleus muscles, was identified. This cDNA was cloned and identified as corresponding to the mRNA of the muscle LIM protein (MLP). Its differential expression in control, stimulated TA, and soleus muscles was verified by Northern blotting. Antibodies against MLP were used to identify by immunoblot analysis a protein of 22 kDa, the predicted molecular mass of MLP. Immunohistochemistry revealed strong reactivity for MLP in all fibers of normal soleus muscle and faint staining of some type IIA and type I fibers in control TA muscle. These fibers increased in number and staining intensity in 4-day-stimulated TA muscle. MLP thus seems to play an essential role during the rearrangement of cytoskeletal and/or myofibrillar structures in transforming adult muscle fibers. (+info)
Ajuba, a novel LIM protein, interacts with Grb2, augments mitogen-activated protein kinase activity in fibroblasts, and promotes meiotic maturation of Xenopus oocytes in a Grb2- and Ras-dependent manner.
LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner. (+info)
Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
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)