The S100A4 metastasis factor regulates cellular motility via a direct interaction with myosin-IIA.

S100A4, a member of the Ca(2+)-dependent S100 family of proteins, is a metastasis factor that is thought to regulate the motility and invasiveness of cancer cells. Previously, we showed that S100A4 specifically binds to nonmuscle myosin-IIA and promotes the unassembled state. S100A4, thus, provides a connection between the actomyosin cytoskeleton and the regulation of cellular motility; however, the step or steps in the motility cycle that are affected by S100A4 expression have not been investigated. To examine how the biochemical properties of S100A4 affect cell motility, we determined the effect of S100A4 expression on protrusive behavior during chemoattractant-stimulated motility. Our studies show that S100A4 modulates cellular motility by affecting cell polarization, with S100A4 expressing cells displaying few side protrusions and extensive forward protrusions during chemotaxis compared with control cells. To establish a direct link between S100A4 and the regulation of myosin-IIA function, we prepared an antibody to the S100A4 binding site on the myosin-IIA heavy chain that has comparable effects on myosin-IIA assembly as S100A4. Microinjection experiments show that the antibody elicits the same effects on cell polarization as S100A4. Our studies show for the first time that S100A4 promotes directional motility via a direct interaction with myosin-IIA. These findings establish S100A4 as a critical regulator of myosin-II function and metastasis-associated motility.

[1]  H. Gaylord,et al.  AMERICAN ASSOCIATION FOR CANCER RESEARCH. , 1913, California state journal of medicine.

[2]  B. Moore.  A soluble protein characteristic of the nervous system. , 1965, Biochemical and biophysical research communications.

[3]  S. Perry,et al.  An electrophoretic study of the low-molecular-weight components of myosin. , 1970, The Biochemical journal.

[4]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[5]  J. Spudich,et al.  The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. , 1971, The Journal of biological chemistry.

[6]  E. Reisler.  Kinetic studies with synthetic myosin minifilaments show the equivalence of actomyosin and acto-HMM ATPases. , 1980, The Journal of biological chemistry.

[7]  G. Nicolson,et al.  Development and biologic properties of malignant cell sublines and clones of a spontaneously metastasizing rat mammary adenocarcinoma. , 1982, Journal of the National Cancer Institute.

[8]  E. Reisler,et al.  The actomyosin ATPase of synthetic myosin minifilaments, filaments, and heavy meromyosin. , 1983, The Journal of biological chemistry.

[9]  G. Nicolson,et al.  Comparison of 'spontaneous' and 'experimental' metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones. , 1983, Invasion & metastasis.

[10]  B. Jockusch,et al.  Modulation of cellular morphology and locomotory activity by antibodies against myosin , 1988, The Journal of cell biology.

[11]  M. Grigorian,et al.  Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family. , 1989, Genes & development.

[12]  F S Fay,et al.  Calcium gradients underlying polarization and chemotaxis of eosinophils. , 1991, Science.

[13]  G. Côté,et al.  Actin-activated Mg-ATPase activity of Dictyostelium myosin II. Effects of filament formation and heavy chain phosphorylation. , 1992, The Journal of biological chemistry.

[14]  J. Daniel,et al.  Purification and characterization of platelet myosin. , 1992, Methods in enzymology.

[15]  D. Lansing Taylor,et al.  Patterns of elevated free calcium and calmodulin activation in living cells , 1992, Nature.

[16]  K. Kohama,et al.  Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene , 1993, FEBS letters.

[17]  P. Rudland,et al.  Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1. , 1993, Oncogene.

[18]  L. Liotta,et al.  Tumor cell interactions with the extracellular matrix during invasion and metastasis. , 1993, Annual review of cell biology.

[19]  Y. Hasegawa,et al.  Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin , 1994, The Journal of cell biology.

[20]  Yohko Nakamura,et al.  Involvement of Sl00–related Calcium–binding Protein pEL98 (or mts1) in Cell Motility and Tumor Cell Invasion , 1994, Japanese journal of cancer research : Gann.

[21]  T. Pollard,et al.  Differential localization of myosin-II isozymes in human cultured cells and blood cells. , 1994, Journal of cell science.

[22]  D. Zimmer,et al.  The S100 protein family: History, function, and expression , 1995, Brain Research Bulletin.

[23]  M. Grigorian,et al.  Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. , 1996, Oncogene.

[24]  E. Hovig,et al.  Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme. , 1996, Cancer research.

[25]  P. Rudland,et al.  Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours. , 1996, Oncogene.

[26]  M. Grigorian,et al.  Effect of mts1 (S100A4) expression on the progression of human breast cancer cells , 1996, International journal of cancer.

[27]  Yohko Nakamura,et al.  Expression of antisense RNA to S100A4 gene encoding an S100-related calcium-binding protein suppresses metastatic potential of high-metastatic Lewis lung carcinoma cells , 1997, Oncogene.

[28]  H. Ford,et al.  Effect of Mts1 on the structure and activity of nonmuscle myosin II. , 1997, Biochemistry.

[29]  R. Kalluri,et al.  Early role of Fsp1 in epithelial-mesenchymal transformation. , 1997, American journal of physiology. Renal physiology.

[30]  I. Bronstein,et al.  Metastasis-associated Mts1 (S100A4) Protein Modulates Protein Kinase C Phosphorylation of the Heavy Chain of Nonmuscle Myosin* , 1998, The Journal of Biological Chemistry.

[31]  R. Carraway,et al.  Signaling pathways underlying eosinophil cell motility revealed by using caged peptides. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  T. Yanagida,et al.  Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[33]  G P Nolan,et al.  High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. , 1998, Cancer research.

[34]  J H Hartwig,et al.  Gelsolin is a downstream effector of rac for fibroblast motility , 1998, The EMBO journal.

[35]  O. T. Odegaard,et al.  S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme. , 1999, Cancer research.

[36]  J. Segall,et al.  A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. , 2000, Cancer research.

[37]  G. Selivanova,et al.  Tumor Suppressor p53 Protein Is a New Target for the Metastasis-associated Mts1/S100A4 Protein , 2001, The Journal of Biological Chemistry.

[38]  L. Mazzucchelli.  Protein S100A4: too long overlooked by pathologists? , 2002, The American journal of pathology.

[39]  E. Neilson,et al.  The gatekeeper effect of epithelial-mesenchymal transition regulates the frequency of breast cancer metastasis. , 2003, Cancer research.

[40]  Howard R. Petty,et al.  Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study1 , 2003, The Journal of Immunology.

[41]  D. Helfman,et al.  Characterization of the Metastasis-associated Protein, S100A4 , 2003, Journal of Biological Chemistry.

[42]  A. Bresnick,et al.  Mts1 regulates the assembly of nonmuscle myosin-IIA. , 2003, Biochemistry.

[43]  J. Condeelis,et al.  Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation , 2004, The Journal of cell biology.

[44]  J. Pollard,et al.  A Paracrine Loop between Tumor Cells and Macrophages Is Required for Tumor Cell Migration in Mammary Tumors , 2004, Cancer Research.

[45]  C. Heizmann,et al.  S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). , 2004, Biochemical and biophysical research communications.

[46]  Fumio Matsumura,et al.  Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts , 2004, The Journal of cell biology.

[47]  H. Petty,et al.  Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells , 2004, Cancer Research.

[48]  J. Kendrick‐Jones,et al.  Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies , 1988, Journal of Muscle Research & Cell Motility.

[49]  Erik Sahai,et al.  Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. , 2005, Cancer research.

[50]  V. Malashkevich,et al.  Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation. , 2005, Biochemistry.

[51]  D. Helfman,et al.  The metastasis associated protein S100A4: role in tumour progression and metastasis , 2005, British Journal of Cancer.