Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.     

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.     

Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.     

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells. J. Cell. Biochem. 68:389–401, 1998. © 1998 Wiley-Liss, Inc.     

Coexpression and nuclear colocalization of metastasis-promoting protein S100A4 and p53 without mutual regulation in colorectal carcinoma

Nuclear localization of the metastasis-associated protein S100A4 has been shown to correlate with advanced disease stage in primary colorectal carcinomas (CRC), but nuclear function and its relevance for the metastatic capacity of tumor cells is still unclear. Among several nuclear interacting protein partners suggested for S100A4, the tumor suppressor protein p53 has attracted particular interest, and previous studies suggest direct and indirect modes of interaction between the two proteins. The present study was undertaken to assess coexpression and potential interaction in CRC. TP53 mutational status and S100A4 expression were investigated in a selected series of primary CRC specimens (n = 40) and cell lines (n = 17) using DNA sequencing, western blot, and double immunostaining. Additionally, S100A4 and p53 were experimentally up- and down-regulated in vitro to assess reciprocal effects. For the first time, S100A4 and p53 coexpression was demonstrated in individual CRC cells, with nuclear colocalization as a particularly interesting feature. In contrast to previous studies, no correlation was observed between TP53 mutational status and S100A4 expression, and no evidence was obtained to support reciprocal regulation between the two molecules in the HCT116 isogenic cell line model. In conclusion, S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells, and it might be speculated whether the proteins interact in this subcellular compartment.     

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.     

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.     

Up-regulation, modification, and translocation of S100A6 induced by exposure to ionizing radiation revealed by proteomics profiling

The cellular response to genotoxic stress is a complex cascade of events including altered protein expression, interactions, modifications, and relocalization, leading to cell cycle arrest and DNA repair or to apoptosis. p53 protein has a central role in this process, and p53 status is an important factor in the response of a tumor to genotoxic anticancer therapy. We studied p53-related changes postexposure to ionizing radiation using top-down mass spectrometry. Initially two cell lines were compared, HCT116 p53 wild type (wt) and p53(-/-), in a time course study postirradiation. In the p53 wt cell line a striking increase of a 10.2-kDa protein was detected, and this protein was identified with MS/MS analysis as S100A6. Further MS profiling led to detection of two post-translationally modified variants of S100A6, namely glutathionylated and cysteinylated forms. In p53 wt cells, a specific shift from glutathionylated to cysteinylated S100A6 occurred postirradiation. The p53 dependence of this specific change in protein level and modification pattern of S100A6 postirradiation was confirmed in a panel of four lung cancer cell lines (H23, U1810, H69, and A549) with different p53 status and using small interfering RNA against p53. Interestingly the closely related S100 family protein S100A4 showed the same changes in modification pattern post-ionizing radiation in the p53 wt lung cancer cell line, and S100A4 also showed p53-dependent expression. Using confocal microscopy, relocalization of S100A6 from nucleus to cytosol and a colocalization with tropomyosin in stress fibers was detected in A549 cells postirradiation. This relocalization coincided with the change in S100A6 modification pattern. Based on these results, we suggest that S100A6 and S100A4 are regulated via redox modifications in vivo and that these proteins are involved in the cellular response to genotoxic stress.     

S100A6 Competes with the TAZ2 Domain of p300 for Binding to p53 and Attenuates p53 Acetylation

S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation.     

Effects of the regulatory light chain phosphorylation of myosin II on mitosis and cytokinesis of mammalian cells

Myosin plays an important role in mitosis, especially during cytokinesis. Although it has been assumed that phosphorylation of regulatory light chain of myosin (RLC) controls motility of mammalian non-muscle cells, the functional significance of RLC phosphorylation remains uninvestigated. To address this problem, we have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal cleavage furrows, resulting in incomplete cytokinesis, which suggests that myosin phosphorylation is important for the normal recruitment of myosin molecules into the contractile ring structure. The other is that separation of chromosomes from the metaphase plate is disrupted in T18A/S19A RLC expressing cells, thus preventing proper transition from metaphase to anaphase. These results suggest that, in addition to cytokinesis, myosin and myosin phosphorylation play a role in the karyokinetic process.     

Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.     

Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 °C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.     

Posttranslational Modifications Affect the Interaction of S100 Proteins with Tumor Suppressor p53

Proteins of the S100 family bind to the intrinsically disordered transactivation domain (TAD; residues 1-57) and C-terminus (residues 293-393) of the tumor suppressor p53. Both regions provide sites that are subject to posttranslational modifications, such as phosphorylation and acetylation, that can alter the affinity for interacting proteins such as p300 and MDM2. Here, we found that S100A1, S100A2, S100A4, S100A6, and S100B bound to two subdomains of the TAD (TAD1 and TAD2). Both subdomains were mandatory for high-affinity binding to S100 proteins. Phosphorylation of Ser and Thr residues increased the affinity for the p53 TAD. Conversely, acetylation and phosphorylation of the C-terminus of p53 decreased the affinity for S100A2 and S100B. In contrast, we found that nitrosylation of S100B caused a minor increase in binding to the p53 C-terminus, whereas binding to the TAD remained unaffected. As activation of p53 is usually accompanied by phosphorylation and acetylation at several sites, our results suggest that a shift in binding from the C-terminus in favor of the N-terminus occurs upon the modification of p53. We propose that binding to the p53 TAD might be involved in the stimulation of p53 activity by S100 proteins.     

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.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.