Liprin beta 1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, is a new target for the metastasis-associated protein S100A4 (Mts1)

Metastasis-associated protein S100A4 (Mts1) induces invasiveness of primary tumors and promotes metastasis. S100A4 belongs to the family of small calcium-binding S100 proteins that are involved in different cellular processes as transducers of calcium signal. S100A4 modulates properties of tumor cells via interaction with its intracellular targets, heavy chain of non-muscle myosin and p53. Here we report identification of a new molecular target of the S100A4 protein, liprin beta1. Liprin beta1 belongs to the family of leukocyte common antigen-related (LAR) transmembrane tyrosine phosphatase-interacting proteins that may regulate LAR protein properties via interaction with another member of the family, liprin alpha1. We showed by the immunoprecipitation analysis that S100A4 interacts specifically with liprin beta1 in vivo. Immunofluorescence staining demonstrated the co-localization of S100A4 and liprin beta1 in the cytoplasm and particularly at the protrusion sites of the plasma membrane. We mapped the S100A4 binding site at the C terminus of the liprin beta1 molecule between amino acid residues 938 and 1005. The S100A4-binding region contains two putative phosphorylation sites by protein kinase C and protein kinase CK2. S100A4-liprin beta1 interaction resulted in the inhibition of liprin beta1 phosphorylation by both kinases in vitro.     

Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form

S100A4 (metastasin) is a member of the S100 family of calcium-binding proteins that is directly involved in tumorigenesis. Until recently, the only structural information available was the solution NMR structure of the inactive calcium-free form of the protein. Here we report the crystal structure of human S100A4 in the active calcium-bound state at 2.03 Å resolution that was solved by molecular replacement in the space group P6₅ with two molecules in the asymmetric unit from perfectly merohedrally twinned crystals. The Ca^2 +-bound S100A4 structure reveals a large conformational change in the three-dimensional structure of the dimeric S100A4 protein upon calcium binding. This calcium-dependent conformational change opens up a hydrophobic binding pocket that is capable of binding to target proteins such as annexin A2, the tumor-suppressor protein p53 and myosin IIA. The structure of the active form of S100A4 provides insight into its interactions with its binding partners and a better understanding of its role in metastasis.     

Evaluation of potential interactions between the metastasis-associated protein S100A4 and the tumor suppressor protein p53

Metastasis is a complex cascade of events involving a finely tuned interplay between malignant cells and multiple host factors. The transition from benign tumor growth to malignancy is manifested by the ability of tumor cells to traverse tissue barriers and invade surrounding tissues. Among a multitude of factors playing a role, the small calcium-binding protein S100A4 has been found to add to the invasive and metastatic capacity of cancer cells. However, the exact molecular function or mechanism by which S100A4 exerts its putative metastasis-promoting effects has not been fully elucidated, and the protein is most likely involved in several aspects of tumor progression. Several studies have recently described a direct interaction and/or reciprocal influence between S100A4 and the tumor suppressor protein p53. This corresponds to reports linking p53 to other S100-family members, especially S100B. The consequences are intriguing, connecting the metastasis-promoting protein S100A4 to the large set of important p53-mediated functions, with broad potential importance in cancer development and metastasis. In this review we emphasize the studies involving p53 and S100A4, elucidating and comparing reported results and conclusions.     

Crystal structure of the Ca(2+)-form and Ca(2+)-binding kinetics of metastasis-associated protein, S100A4

S100A4 takes part in control of tumour cell migration and contributes to metastatic spread in in vivo models. In the active dimeric Ca(2+)-bound state it interacts with multiple intracellular targets. Conversely, oligomeric forms of S100A4 are linked with the extracellular function of this protein. We report the 1.5A X-ray crystal structure of Ca(2+)-bound S100A4 and use it to identify the residues involved in target recognition and to derive a model of the oligomeric state. We applied stopped-flow analysis of tyrosine fluorescence to derive kinetics of S100A4 activation by Ca(2+) (k(on)=3.5 microM(-1)s(-1), k(off)=20s(-1)).     

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

Hsp70 is a new target of Sgt1--an interaction modulated by S100A6

Here, two temperature sensitive promoters, P2 and P7, isolated from Bacillus subtilis, were characterized. The production of beta-galactosidase driven by these promoters was much higher at 45 degrees C than that at 37 degrees C both in Escherichia coli and B. subtilis and that the P2 promoter showed higher expression strength in B. subtilis at 45 degrees C. Thereby, an efficient temperature-inducible expression system was constructed by using P2 promoter in B. subtilis. Thus, we isolated and characterized a newly temperature inducible promoter and exploited it as a potential expression element in B. subtilis.     

Characterization of the metastasis-associated protein,S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin

Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimers and interacts with several proteins in a calcium-dependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancer-derived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calcium-independent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.     

The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies

The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.     

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.     

Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition

S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.     

Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan

Staphylococcus aureus aminopeptidase S (AmpS) has been named for its predicted, but experimentally untested, aminopeptidase activity. The enzyme is homologous to biochemically characterized aminopeptidases that contain two cobalt or zinc ions in their active centers, but it is unrelated to all structurally characterized metallopeptidases. Here, we demonstrate AmpS aminopeptidase activity experimentally, and we present the 1.8-A crystal structure of the enzyme. Two metal ions with full occupancy and a third metal ion with low occupancy are present in the active site. A water molecule and Glu-319 serve as bridging ligands to the two metals with full occupancy. One of these metal ions is additionally coordinated by Glu-253 and His-348 and the other by His-381 and Asp-383. In addition, the metals are involved in weak metal-donor interactions to a water molecule and to Tyr-355. In the crystal, AmpS forms a dimer with a large internal cavity. The active sites are located at opposite ends of this internal cavity and are essentially inaccessible from the outside, suggesting that an inactive conformation was crystallized. Because gel filtration and analytical ultracentrifugation data also suggest dimer formation, the problem of substrate access to the active site cavity remains unresolved.     

HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein

The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.     

Methionine aminopeptidase gene of Escherichia coli is essential for cell growth.

We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-beta-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.     

S100A1, a new marker for acute myocardial ischemia

The Ca(2+)-binding S100A1 protein displays a specific and differential expression in the human myocardium and is considered to be an important regulator of heart function. Because of its high expression levels in the heart we tested the performance of S100A1 as a diagnostic indicator of acute myocardial damage. Therefore, we established a S100A1-specific sandwich ELISA and determined the S100A1 plasma levels in patients with signs of acute myocardial ischemia. The concentration-time course of S100A1 was distinct from that of the "classical" biochemical markers, CK, CKMB, and troponin I, showing an early rise and a fast decline in plasma after the ischemic event. We suggest that S100A1 should be included in combinatorial measurements as an early diagnostic marker for ischemic coronary diseases.     

Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.     

The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.