An interaction between zyxin and alpha-actinin

Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein alpha-actinin. Zyxin cosediments with filamentous actin in an alpha-actinin-dependent manner and an association between zyxin and alpha-actinin is observed in solution by analytical gel filtration. The specificity of the interaction between zyxin and alpha-actinin was demonstrated by blot overlay experiments in which 125I-zyxin recognizes most prominently alpha-actinin among a complex mixture of proteins extracted from avian smooth muscle. By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal 27-kD domain of alpha-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxin-alpha-actinin interaction. By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and alpha-actinin. Furthermore, double-label immunofluorescence reveals that zyxin and alpha-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells. The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and alpha-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions.     

Analysis of the alpha-actinin/zyxin interaction.

The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin. The zyxin/alpha-actinin interaction was analyzed in detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding.     

The subcellular localization and protein stability of mouse alpha-actinin 2 is controlled by its nuclear receptor binding motif in C2C12 cells

Alpha actinin (ACTN) has emerged as a multitasking protein, whose roles range from bundling actin filaments to functioning as a versatile protein interaction platform for proteins involved in structural or signaling aspects. We report here that ACTN2, one of the four ACTN isoforms, may shuttle between the cytoplasm and nucleus where the nuclear exportation takes place in a CRM1-dependent manner. The majority of ACTN2 was found to localize in the cytoplasm and exhibit a lower stability which was demonstrated using either mutants carrying mutated nuclear receptor binding motif or inhibitors against the ubiquitin- and calpain-dependent degradation pathways. Horse serum induced differentiation of C2C12 cells also caused the redistribution of nuclear ACTN2 to the cytoplasm, which subcellular compartment the ACTN2 behaves as an unstable protein. Our data indicated that the model in which ACTN2 functions as a multi-talented coregulator may be controlled by the differential protein stability modulated via nucleo-cytoplasmic trafficking in C2C12 cells.     

Phosphatidylserine binding is essential for plasma membrane recruitment and signaling function of 3-phosphoinositide-dependent kinase-1

3-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3'-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1.     

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs (¹⁴¹FFVKLYFTF¹⁴⁹ and ²⁹⁹YDFPEKFF³⁰⁶) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant (¹⁴¹FFVKLYFTF¹⁴⁹ and ²⁹⁹YDFPEKFF³⁰⁶ change to ¹⁴¹AFVKLAFTA¹⁴⁹ and ²⁹⁹ADAPEFLA³⁰⁶) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.     

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.     

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named α-synemin and β-synemin, with the larger α-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long tail domain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.     

The LIM domain of zyxin is sufficient for force-induced accumulation of zyxin during cell migration

Cellular responses to mechanical perturbation are vital to cell physiology. In particular, migrating cells have been shown to sense substrate stiffness and alter cell morphology and speed. Zyxin is a focal adhesion protein that responds to external mechanical forces; however, the mechanisms of zyxin recruitment at force-bearing sites are unknown. Using force-sensing microfabricated substrates, we simultaneously measured traction force and zyxin recruitment at force-bearing sites. GFP-tagged zyxin accumulates at force-bearing sites at the leading edge, but not at the trailing edge, of migrating epithelial cells. Zyxin recruitment at force-bearing sites depends on Rho-kinase and myosin II activation, suggesting that zyxin responds not only to the externally applied force, as previously shown, but also to the internally generated actin-myosin force. Zyxin in turn recruits vasodilator-stimulated phosphoprotein, a regulator of actin assembly, to force-bearing sites. To dissect the domains of zyxin that are essential for this unique force-dependent accumulation, we generated two zyxin truncation mutants: one lacking the LIM domain (ΔLIM) and one containing only the LIM domain with all three LIM motifs (LIM). GFP-tagged ΔLIM does not localize to the force-bearing sites, but GFP-tagged zyxin LIM-domain is sufficient for the recruitment to and dynamics at force-bearing focal adhesions. Furthermore, one or two LIM motifs are not sufficient for force-dependent accumulation, suggesting that all three LIM motifs are required. Therefore, the LIM domain of zyxin recruits zyxin to force-bearing sites at the leading edge of migrating cells.     

A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms.     

Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy

Cell-matrix adhesions in migrating cells are usually mediated by integrins, alpha-beta heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the beta1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle alpha-actinin. The 2-D arrays of the beta1-integrin-alpha-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled beta1-integrin peptide. The difference maps indicate that the beta1-integrin cytoplasmic domain binds alpha-actinin between the first and second, 3-helix motifs in the central rod domain.     

ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

Z-DNA binding protein 1 (ZBP1) belongs to a family of proteins that contain the Zα domain, which binds specifically to left-handed Z-DNA and Z-RNA. Like all vertebrate proteins in the Zα family, it contains two Zα-like domains and is highly inducible by immunostimulation. Using circular dichroism spectroscopy and electrophoretic mobility shift assays we show that both Zα domains can bind Z-DNA independently and that substrate binding is greatly enhanced when both domains are linked. Full length ZBP1 and a prominent splice variant lacking the first Zα domain (ΔZα) showed strikingly different subcellular localizations. While the full length protein showed a finely punctate cytoplasmatic distribution, ZBP1ΔZα accumulated in large cytoplasmic granules. Mutation of residues important for Z-DNA binding in the first Zα domain resulted in a distribution comparable to that of ZBP1ΔZα. The ZBP1ΔZα granules are distinct from stress granules (SGs) and processing bodies but dynamically interacted with these. Polysome stabilization led to the disassembly of ZBP1ΔZα granules, indicating that mRNA are integral components. Heat shock and arsenite exposure had opposing effects on ZBP1 isoforms: while ZBP1ΔZα granules disassembled, full length ZBP1 accumulated in SGs. Our data link ZBP1 to mRNA sorting and metabolism and indicate distinct roles for ZBP1 isoforms.     

Prediction of essential proteins based on subcellular localization and gene expression correlation

Background

Essential proteins are indispensable to the survival and development process of living organisms. To understand the functional mechanisms of essential proteins, which can be applied to the analysis of disease and design of drugs, it is important to identify essential proteins from a set of proteins first. As traditional experimental methods designed to test out essential proteins are usually expensive and laborious, computational methods, which utilize biological and topological features of proteins, have attracted more attention in recent years. Protein-protein interaction networks, together with other biological data, have been explored to improve the performance of essential protein prediction.

Results

The proposed method SCP is evaluated on Saccharomyces cerevisiae datasets and compared with five other methods. The results show that our method SCP outperforms the other five methods in terms of accuracy of essential protein prediction.

Conclusions

In this paper, we propose a novel algorithm named SCP, which combines the ranking by a modified PageRank algorithm based on subcellular compartments information, with the ranking by Pearson correlation coefficient (PCC) calculated from gene expression data. Experiments show that subcellular localization information is promising in boosting essential protein prediction.

     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.     

Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.

alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.     

Predicting essential proteins based on subcellular localization,orthology and PPI networks

Background

Essential proteins play an indispensable role in the cellular survival and development. There have been a series of biological experimental methods for finding essential proteins; however they are time-consuming, expensive and inefficient. In order to overcome the shortcomings of biological experimental methods, many computational methods have been proposed to predict essential proteins. The computational methods can be roughly divided into two categories, the topology-based methods and the sequence-based ones. The former use the topological features of protein-protein interaction (PPI) networks while the latter use the sequence features of proteins to predict essential proteins. Nevertheless, it is still challenging to improve the prediction accuracy of the computational methods.

Results

Comparing with nonessential proteins, essential proteins appear more frequently in certain subcellular locations and their evolution more conservative. By integrating the information of subcellular localization, orthologous proteins and PPI networks, we propose a novel essential protein prediction method, named SON, in this study. The experimental results on S.cerevisiae data show that the prediction accuracy of SON clearly exceeds that of nine competing methods: DC, BC, IC, CC, SC, EC, NC, PeC and ION.

Conclusions

We demonstrate that, by integrating the information of subcellular localization, orthologous proteins with PPI networks, the accuracy of predicting essential proteins can be improved. Our proposed method SON is effective for predicting essential proteins.

     

An essential arginine residue at the substrate binding site of 4-hydroxyisophthalate hydroxylase

4-Hydroxyisophthalate hydroxylase was inactivated by treatment with phenylglyoxal by a process obeying pseudo-first order kinetics indicating the presence of an essential arginine located presumably in the active site. Addition of saturating amounts of 4-hydroxyisophthalate during the treatment resulted in complete protection of the enzyme from the inactivation, but addition of NADPH was totally ineffective. Analysis of the effect of various substrate analogs on the protection of the enzyme showed that carboxyl and hydroxyl groups at para positions on the aromatic ring are essential for substrate binding to the active site. It was also observed that analogs which protect the enzyme against phenylglyoxal inactivation are themselves effective inhibitors of the enzyme activity.     

Low-molecular RNA subcellular localization and its binding strength with cellular structures

Some fractions of low molecular weight (LMW) nuclear RNAs were shown to be present in the cytoplasm of rat liver cells. In addition to known 4S tRNA, 5S and 5,8S rRNAs U3 and 8S1 LMW nuclear RNAs, 8SII and 8SIII LMW RNAs have been detected in RNA preparations of free total and membrane-bound polysomes. The U3 and 8SI polysoma I RNAs seem to be associated with high molecular weight polysomal RNA. Using thermal phenol fractionation, that some LMW RNAs were shown to be slightly bound to the cellular structures whereas some others are bound more tightly. Considerable amounts of LMW RNAs are tightly bound to the chromosome-nucleolar apparatus. They can be extracted only at 85 degrees C. The data presented are discussed with regard to LMW nuclear and polysomal RNAs functions.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.