Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

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.     

The PDZ-LIM protein CLP36 is required for actin stress fiber formation and focal adhesion assembly in BeWo cells

CLP36 belongs to the ALP subfamily of PDZ-LIM proteins and has a PDZ domain at its N-terminal and a LIM domain at its C-terminal. It has been shown that CLP36 is localized to stress fibers through interaction with α-actinin, but its function is still unclear. To investigate the role of CLP36 in stress fibers, we suppressed CLP36 expression in BeWo cells by RNAi and examined the phenotypic changes. CLP36-knockdown resulted in cell spreading and the loss of stress fibers and focal adhesions. These changes were reversed by addition of exogenous CLP36, but not by addition of mutant forms of CLP36 that lacked the PDZ or LIM domain. These findings indicate that CLP36 plays a critical role in stress fiber formation and the assembly of focal adhesions in BeWo cells. In addition, the PDZ and LIM domains are both essential for CLP36 to function.     

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.     

P1 ParB Domain Structure Includes Two Independent Multimerization Domains

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.     

ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.     

Cerivastatin suppresses lipopolysaccharide-induced ICAM-1 expression through inhibition of Rho GTPase in BAEC

Both LIM15/DMC1 and RAD51 are thought to be essential for meiosis in which homologous chromosomes pair and recombine. The primary purpose of the present study was to investigate the homotypic and heterotypic interactions among their terminal domains. We prepared cDNAs and recombinant proteins of the full-length, N-terminal, and the C-terminal domains of LIM15/DMC1 (CoLIM15) and RAD51 (CoRAD51) from the basidiomycete Coprinus cinereus. In both two-hybrid assay in vivo and pull-down assay in vitro, either CoLim15 or CoRad51 interacted homotypically between the C-terminal domains, respectively, but no heterotypic interaction was observed between CoLim15 and CoRad51. The N-terminal domain of CoLim15 bound to ssDNA and dsDNA, while the C-terminal domain of CoRad51 appeared to interact weakly with ssDNA. Based on these results, the interaction among the strand-exchange proteins and meiosis was discussed.     

A zyxin head-tail interaction regulates zyxin-VASP complex formation

Zyxin is an adhesion protein that regulates actin assembly by binding to VASP family members through N-terminal proline-rich motifs. Evidence suggests that zyxin’s C-terminal LIM domains function as a negative regulator of zyxin-VASP complexes. Zyxin LIM domains access to binding partners is negatively regulated by an unknown mechanism. One possibility is that zyxin LIM domains mediate a head-tail interaction, blocking interactions with other proteins. Such a mechanism might prevent both zyxin-VASP complexes activity and LIM domain access. In this report, the effect of LIM domains on zyxin-VASP complex assembly is defined. We find that zyxin LIM domains associate with zyxin’s VASP binding sites, preventing zyxin from binding to PKA-phosphorylated VASP. Unphosphorylated VASP overcomes the head-tail interaction, a result of a direct interaction with the LIM domain region. Zyxin, like a growing number of actin regulators, is controlled by intramolecular interactions.     

Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton

Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1. Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1-positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them. Immunogold double-labeling electron microscopy confirmed that caveolin-1-labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1-associated membrane domains.     

The focal adhesion and nuclear targeting capacity of the LIM-containing lipoma-preferred partner (LPP) protein

Targeting of proteins to a particular cellular compartment is a critical determinant for proper functioning. LPP (LIM-containing lipoma-preferred partner) is a LIM domain protein that is localized at sites of cell adhesion and transiently in the nucleus. In various benign and malignant tumors, LPP is present in a mutant form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its subcellular locations. We found that the LIM domains are the main focal adhesion targeting elements and that the proline-rich region of LPP, which harbors binding sites for alpha-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM domains of LPP cooperate in order to provide robust targeting to focal adhesions, and the linker between LIM domains 1 and 2 plays a pivotal role in this targeting. When overexpressed in the cytoplasm of cells, the LIM domains of LPP can deplete endogenous LPP and vinculin from focal adhesions. The proline-rich region of LPP contains targeting sites for focal adhesions and stress fibers that are distinct from the alpha-actinin and VASP binding sites, and the LPP LIM domains are dispensable for targeting LPP to the nucleus. Our studies have defined novel functional domains in the LPP protein.     

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.     

Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin, while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate that activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin’s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton’s mechanical state.     

Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain.

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.     

A critical role of the PINCH-integrin-linked kinase interaction in the regulation of cell shape change and migration.

The interaction of cells with extracellular matrix recruits multiple proteins to cell-matrix contact sites (e.g. focal and fibrillar adhesions), which connect the extracellular matrix to the actin cytoskeleton and regulate cell shape change, migration, and other cellular processes. We previously identified PINCH, an adaptor protein comprising primarily five LIM domains, as a binding protein for integrin-linked kinase (ILK). In this study, we show that PINCH co-localizes with ILK in both focal adhesions and fibrillar adhesions. Furthermore, we have investigated the molecular basis underlying the targeting of PINCH to the cell-matrix contact sites and the functional significance of the PINCH-ILK interaction. We have found that the N-terminal LIM1 domain, which mediates the ILK binding, is required for the targeting of PINCH to the cell-matrix contact sites. The C-terminal LIM domains, although not absolutely required, play an important regulatory role in the localization of PINCH to cell-matrix contact sites. Inhibition of the PINCH-ILK interaction, either by overexpression of a PINCH N-terminal fragment containing the ILK-binding LIM1 domain or by overexpression of an ILK N-terminal fragment containing the PINCH-binding ankyrin domain, retarded cell spreading, and reduced cell motility. These results suggest that PINCH, through its interaction with ILK, is crucially involved in the regulation of cell shape change and motility.     

Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.     

Actinin-associated LIM protein-deficient mice maintain normal development and structure of skeletal muscle

The actinin-associated LIM protein, ALP, is the prototype of a large family of proteins containing an N-terminal PDZ domain and a C-terminal LIM domain. These PDZ-LIM proteins are components of the muscle cytoskeleton and occur along the Z lines owing to interaction of the PDZ domain with the spectrin-like repeats of alpha-actinin. Because PDZ and LIM domains are typically found in proteins that mediate cellular signaling, PDZ-LIM proteins are suspected to participate in muscle development. Interestingly the ALP gene occurs at 4q35 near the heterochromatic region mutated in facioscapulohumeral muscular dystrophy, indicating a possible role for ALP in this disease. Here, we describe the generation and analysis of mice lacking the ALP gene. Surprisingly, the ALP knockout mice show no gross histological abnormalities and maintain sarcolemmal integrity as determined by serum pyruvate kinase assays. The absence of a dystrophic phenotype in these mice suggests that down-regulation of ALP does not participate in facioscapulohumeral muscular dystrophy. These data suggest that ALP does not participate in muscle development or that an alternative PDZ-LIM protein can compensate for the lack of ALP.     

A 13-amino acid n-terminal domain of a basic proline-rich protein is necessary for storage in secretory granules and facilitates exit from the endoplasmic reticulum.

We have investigated the role of different domains of a salivary basic proline-rich protein in intracellular transport and sorting of proline-rich proteins to the secretory granules. We have cloned a full-length cDNA of a basic proline-rich protein from the rat parotid and expressed it in AtT-20 cells. It was correctly sorted into secretory granules as shown by EM immunolocalization and by its presence in 8-bromocyclic AMP-stimulated secretion. Deletion of the N-terminal thirteen amino acid domain upstream from the proline-rich domain eliminated storage whereas deletion of the C-terminal 20-amino acid domain downstream from the proline-rich domain had no effect. Intracellular transport of full-length and mutant proline-rich proteins was unusually slow due to slow exit from the endoplasmic reticulum. However, the rate of transport increased with increasing level of expression for the full-length protein and the C-terminal deletion mutant. In contrast, the rate of transport of the N-terminal deletion mutant was independent of the level of expression. These results imply that the N-terminal domain is necessary for both storage and efficient intracellular transport. Moreover, interactions (self-aggregation?) that mediate sorting may begin as early as the endoplasmic reticulum.     

Elfin is expressed during early heart development

Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.     

Genetic analysis of interactions between Gag proteins of Rous sarcoma virus.

The yeast two-hybrid system was used to characterize homomeric interactions between the Gag proteins of Rous sarcoma virus (RSV). The RSV Gag precursor was found to interact strongly with itself and not with various control proteins. The RSV Gag did not interact significantly with Gag proteins of a variety of other retroviruses, including murine leukemia viruses and primate lentiviruses. Deletion analysis suggested that two nonoverlapping regions are independently sufficient to mediate RSV Gag-Gag dimerization. One such region lies near the N terminus and contains p2, p10, and a large N-terminal part of the capsid (CA) domain; the other is localized in the C terminus and includes a small C-terminal portion of CA and the nucleocapsid protein. These interaction domains may play roles in viral assembly.