Regulation of non muscle myosin 2 and by S100 and phosphorylation

Thesis of Ph.D. dissertation

Péter Ecsédi

Doctorate School in Biology, Faculty of Science, Eötvös Loránd University Head of Ph.D. School Prof. Anna Erdei Ph.D., DSc

Structural Biochemistry Doctoral Program Program Director Prof. Mihály Kovács Ph.D., DSc

Supervisor: Prof. László Nyitray Ph.D., DSc Head of Department of Biochemistry

Department of Biochemistry, Institute of Biology Faculty of Science, Eötvös Loránd University Budapest, 2018.

Short Introduction

The Ca2+-binding S100 family

The members of the S100 family are small, dimeric, EF-hand Ca2+-binding proteins (1,2). They have high structural similarities, which is characterized by four α- helices and their interconnections forming the canonical and „pseudo” EF hand motifs (3). The initially closed S100 conformation changes upon Ca2+-binding, which than allows the interaction with partner molecules. They have important pathological roles in tumor formation, metastasis and chronic inflammations like rheumatoid arthritis (2,4). It was found that elevated expression level of S100A4 is usually correlating with the formation of high risk metastatic tumors (5,6). In several studies it was shown that the pathological functions of S100A4 are connected to its interactions with non muscle myosin 2A (NM2A) (7,8), annexin A2 (ANXA2) (9,10) and p53 (11).

Non muscle myosin 2, the most investigated S100A4 partner

Members of the myosin superfamily are motor proteins which use the energy of ATP to generate force. In vertebrates there are three known NM2 paralogs: NM2A, NM2B and NM2C (12-14). NM2 paralogs display 64-80 % sequence identity but they differ in ATPase kinetics, motility rates (15,16), intercellular localization (17,18) and they perform distinctive and overlapping cellular functions (19,20). NM2 paralogs can assemble to form functional filaments in cells. The primary regulatory step of this self- assembly is the phosphorylation of the regulatory light chain (RLC). Phosphorylation by casein kinase 2 (CK2), protein kinase C (PKC) and transient receptor potential melastatin 7 (TRPM7) in or near the tailpiece (21-25) and binding of partner proteins like S100A4 (7,8,26-28), lethal giant larvae (Lgl1) protein (29,30) and S100P (31) to the C-terminus of NM2 were shown to further regulate filament assembly/disassembly.

2

It has been recently demonstrated that NM2A and NM2B can form heterotypic filaments in vivo (32-34). However the effect of S100A4 and C-terminal phosphorylation on mixed filaments was not investigated. The question therefore remains as to whether the equilibrium between heterofilaments and homofilaments can be selectively shifted either by S100A4 binding or C-terminal phosphorylation, representing a novel secondary sorting mechanism of non-muscle myosins.

Annexin A2, an additional S100A4 partner

Annexin A2 (ANXA2) is a member of the non-EF-hand Ca2+-binding protein family of annexins. It has a versatile role in membrane-associated functions including membrane repair and aggregation, endo- and exocytosis. The protein consists of a C- terminal domain (CTD) containing the Ca2+-binding sites (important for membrane binding) on its convex side. N-terminally a short linear sequence is located (NTD) which interacts with the CTD causing its highly conserved localization. ANXA2 functions are known to be regulated by post-translational modifications and protein-protein interactions occurring in the NTD. Several different topological models have been proposed to explain ANXA2-mediated membrane aggregation (PMID:(35-37). However, without the knowledge of any 3D structures for phosphorylated ANXA2, the mechanism of phosphorylation-mediated regulation remains unclear.

Objectives

Over my doctoral years I studied the S100 family, mainly S100A4. The following research questions and objectives were formulated:

 My aim was to examine the phosphorylated NM2 paralogs and ANXA2 to investigate the effect of this modification. Other groups have also carried out similar studies, but in case of myosins the literature contains contradictions, which may be due to the fact that previous model systems have a filament-forming ability other than the full-length proteins. In addition, similarly to the phosphorylation of

3

ANXA2, the mechanism of NM2 phosphorylation is also unknown, due to the lack of structural works in this field.  Based on previous results found by our group examining S100A4-NM2A interaction, I produced all members of the family to study the effects of all S100s on myosin filaments. It was shown that S100P is able to destabilize NM2A filaments, but systematical investigations were not performed. In addition, I aimed to examine how selective those interactions are in case of NM2A and NM2B.  One of my main goals was to map the selective regulation of C-terminal phosphorylation and S100A4 binding on heterofilaments formed by NM2A and NM2B to study the possibility of a sorting mechanism in cell.  My further objective was to examine the binding of S100A4 to ANXA2 in parallel with the long-known S100A10 - ANXA2 interaction. Solving the structure of S100A4 – ANXA2 complex was one of the most prominent elements of my doctoral work. Thereby knowing the stoichiometry of the complex could help us to decide if the asymmetric interaction found in case of NM2A – S100A4 is unique or more general in the S100 family.  I studied the effects of S100 proteins together with C-terminal phosphorylation events on the stability of myosin filaments in order to compare the two types of regulation mechanism and to find out if there is any crossregulation between them.  Finally, I examined the effects of S100A4 and S100A10 on NTD phosphorylated ANXA2. Using these results, my ultimate objective was to suggest a model including events affecting ANXA2 membrane aggregating ability and their mechanism.

Applied Methods

Protein constructs were mostly produced by heterologous expression in bacterial systems. Some of them were also phosphorylated in vitro with kinases also produced in bacterial systems. Protein-protein interactions were studied using biochemical methods 4 such as fluorescence polarization (FP), isothermal titrational calorimetry (ITC), TIRF microscopy, and filament disassembly assay. Structural information were obtained by protein crystallography, NMR, small angle X-ray scattering (SAXS), electron microscopy (EM) and molecular dynamics (MD) simulation.

Theses and discussion

 A fluorescent protein was fused N-terminally to the coiled coil region of NM2A and NM2B. These constructs, similarly to full length myosins, form bipolar filaments under physiological conditions. This is important, because the filament- forming properties of myosin fragments used in previous in vitro studies are different and those form only regular aggregates, called paracrystals1.  The stability of NM2B filaments is more sensitive to C-terminal phosphorylation compared to NM2A. These phosphorylation events have synergistically negative effects on filament formation (dimmer). We suggest that the more sites are phosphorylated at the tail, the C-terminus becomes more negatively charged, shifting filaments toward disassembly by intermolecular repulsions1.  Structural studies show that the stability of ANXA2 and the flexibility of NTD are regulated by NTD phosphorylataion acting as a molecular switch. The modification of Tyr24 keeps ANXA2 in a more stable "closed" conformation and acts as an anchor to clamp the NTD to the CTD through a novel ionic interaction. The effect of Ser26 phosphorylation is contrary since NTD detaches from CTD after this modification shifting ANXA2 structure towards a less stable "open" conformation2.  The phosphorylation of Tyr24 almost completely inhibits membrane aggregation ability of ANXA2. The novel anchoring point that fixes the NTD to the CTD does not loosen after membrane binding inhibiting the association of two ANXA2 molecules via their concave sides. The phosphorylation of Ser26 only partially inhibits this function. Knowing the structure of this variant, it is probable that Ser26 phosphorylation introduces a novel autoinhibited conformation2. 5

 In contrast to phosphorylation events, S100 proteins are selectively capable of disassembling NM2A filaments, while they affect NM2B filaments two orders of magnitude weaker. In addition to the known S100A4 interaction, S100A1, S100A2, S100A6, S100B and S100P also bind to NM2A with high affinity. Based on these results, it would be advisable to monitor not only one member of the family as a biomarker during metastasis or other chronic S100 related diseases, but specific collections of them1.  S100 interaction and C-terminal phosphorylation can selectively disassemble NM2A and NM2B formed heterofilaments in vitro. If these regulatory events also occur inside of the cell, they represent an unexplored level of non-muscle myosin regulation1.  The ANXA2 - S100A10 and ANXA2 - S100A4 interactions have different affinities and stoichiometry. The solved ANXA2 - S100A4 complex structure shows that the asymmetric interaction found in case of NM2A - S100A4 can be more general within the S100 family2.  C-terminal phosphorylation of NM2 paralogs does not affect the binding abilities of S100 proteins1.  The inhibition of ANXA2 membrane aggregation function caused by Tyr24 phosphorylation can be eliminated by S100A4 and S100A10 binding, but with different mechanisms due to their different binding modes2.

Publications used to establish theses

1 Paralog selective regulation of non-muscle myosin 2 filaments by S100 protein binding and C- terminal phosphorylation Péter Ecsédi, Neil Billington, Gyula Pálfy, Gergő Gógl, Bence Kiss, Éva Bulyáki, Andrea Bodor, James R.Sellers and László Nyitray, Structure, 2017. 25 (8): p. 1195-1207. e5

2 Regulation of the equilibrium between closed and open conformations of annexin A2 by N-terminal phosphorylation and S100A4-binding Péter Ecsédi, Bence Kiss, Gergő Gógl, László Radnai, László Buday, Kitti Koprivanacz, Károly Liliom, Ibolya Leveles, Beáta Vértessy, Norbert Jeszenői, Csaba Hetényi, Gitta Schlosser, Gergely Katona and László Nyitray, JBC/2018/004277

6

Referenciák

1. Moore, B. W. (1965) A soluble protein characteristic of the nervous system. Biochemical and biophysical research communications 19, 739- 744 2. Bresnick, A. R., Weber, D. J., and Zimmer, D. B. (2015) S100 proteins in cancer. Nature reviews. Cancer 15, 96-109 3. Donato, R., Cannon, B. R., Sorci, G., Riuzzi, F., Hsu, K., Weber, D. J., and Geczy, C. L. (2013) Functions of S100 proteins. Current molecular medicine 13, 24-57 4. Boye, K., and Maelandsmo, G. M. (2010) S100A4 and metastasis: a small actor playing many roles. The American journal of pathology 176, 528-535 5. Helfman, D. M., Kim, E. J., Lukanidin, E., and Grigorian, M. (2005) The metastasis associated protein S100A4: role in tumour progression and metastasis. British journal of cancer 92, 1955-1958 6. Garrett, S. C., Varney, K. M., Weber, D. J., and Bresnick, A. R. (2006) S100A4, a mediator of metastasis. The Journal of biological chemistry 281, 677-680 7. Kriajevska, M. V., Cardenas, M. N., Grigorian, M. S., Ambartsumian, N. S., Georgiev, G. P., and Lukanidin, E. M. (1994) Non-muscle myosin heavy chain as a possible target for protein encoded by metastasis-related mts-1 . The Journal of biological chemistry 269, 19679- 19682 8. Ford, H. L., Silver, D. L., Kachar, B., Sellers, J. R., and Zain, S. B. (1997) Effect of Mts1 on the structure and activity of nonmuscle myosin II. Biochemistry 36, 16321-16327 9. MacLeod, T. J., Kwon, M., Filipenko, N. R., and Waisman, D. M. (2003) Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin. The Journal of biological chemistry 278, 25577-25584 10. Semov, A., Moreno, M. J., Onichtchenko, A., Abulrob, A., Ball, M., Ekiel, I., Pietrzynski, G., Stanimirovic, D., and Alakhov, V. (2005) Metastasis-associated protein S100A4 induces angiogenesis through interaction with Annexin II and accelerated plasmin formation. The Journal of biological chemistry 280, 20833-20841 11. Orre, L. M., Panizza, E., Kaminskyy, V. O., Vernet, E., Graslund, T., Zhivotovsky, B., and Lehtio, J. (2013) S100A4 interacts with p53 in the nucleus and promotes p53 degradation. Oncogene 32, 5531-5540 12. Katsuragawa, Y., Yanagisawa, M., Inoue, A., and Masaki, T. (1989) Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains. European journal of biochemistry 184, 611-616 13. Golomb, E., Ma, X., Jana, S. S., Preston, Y. A., Kawamoto, S., Shoham, N. G., Goldin, E., Conti, M. A., Sellers, J. R., and Adelstein, R. S. (2004) Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family. The Journal of biological chemistry 279, 2800-2808 14. Simons, M., Wang, M., McBride, O. W., Kawamoto, S., Yamakawa, K., Gdula, D., Adelstein, R. S., and Weir, L. (1991) Human nonmuscle myosin heavy chains are encoded by two located on different . Circulation research 69, 530-539 15. Kovacs, M., Wang, F., Hu, A., Zhang, Y., and Sellers, J. R. (2003) Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform. The Journal of biological chemistry 278, 38132-38140 16. Rosenfeld, S. S., Xing, J., Chen, L. Q., and Sweeney, H. L. (2003) Myosin IIb is unconventionally conventional. The Journal of biological chemistry 278, 27449-27455 17. Saitoh, T., Takemura, S., Ueda, K., Hosoya, H., Nagayama, M., Haga, H., Kawabata, K., Yamagishi, A., and Takahashi, M. (2001) Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts. FEBS letters 509, 365-369 18. Beach, J. R., Hussey, G. S., Miller, T. E., Chaudhury, A., Patel, P., Monslow, J., Zheng, Q., Keri, R. A., Reizes, O., Bresnick, A. R., Howe, P. H., and Egelhoff, T. T. (2011) Myosin II isoform switching mediates invasiveness after TGF-beta-induced epithelial-mesenchymal transition. Proceedings of the National Academy of Sciences of the United States of America 108, 17991-17996 19. Sandquist, J. C., Swenson, K. I., Demali, K. A., Burridge, K., and Means, A. R. (2006) Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration. The Journal of biological chemistry 281, 35873-35883 20. Even-Ram, S., Doyle, A. D., Conti, M. A., Matsumoto, K., Adelstein, R. S., and Yamada, K. M. (2007) Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nature cell biology 9, 299-309 21. Dulyaninova, N. G., Malashkevich, V. N., Almo, S. C., and Bresnick, A. R. (2005) Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation. Biochemistry 44, 6867-6876 22. Murakami, N., Matsumura, S., and Kumon, A. (1984) Purification and identification of myosin heavy chain kinase from bovine brain. Journal of biochemistry 95, 651-660 23. Murakami, N., Chauhan, V. P., and Elzinga, M. (1998) Two nonmuscle myosin II heavy chain isoforms expressed in rabbit brains: filament forming properties, the effects of phosphorylation by protein kinase C and casein kinase II, and location of the phosphorylation sites. Biochemistry 37, 1989-2003 24. Even-Faitelson, L., and Ravid, S. (2006) PAK1 and aPKCzeta regulate myosin II-B phosphorylation: a novel signaling pathway regulating filament assembly. Molecular biology of the cell 17, 2869-2881 25. Clark, K., Middelbeek, J., Lasonder, E., Dulyaninova, N. G., Morrice, N. A., Ryazanov, A. G., Bresnick, A. R., Figdor, C. G., and van Leeuwen, F. N. (2008) TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation. Journal of molecular biology 378, 790-803 26. Murakami, N., Kotula, L., and Hwang, Y. W. (2000) Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA. Biochemistry 39, 11441-11451 27. Kiss, B., Duelli, A., Radnai, L., Kekesi, K. A., Katona, G., and Nyitray, L. (2012) Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism. Proceedings of the National Academy of Sciences of the United States of America 109, 6048-6053 7

28. Li, Z. H., and Bresnick, A. R. (2006) The S100A4 metastasis factor regulates cellular motility via a direct interaction with myosin-IIA. Cancer research 66, 5173-5180 29. Dahan, I., Petrov, D., Cohen-Kfir, E., and Ravid, S. (2014) The tumor suppressor Lgl1 forms discrete complexes with NMII-A and Par6alpha- aPKCzeta that are affected by Lgl1 phosphorylation. Journal of cell science 127, 295-304 30. Dahan, I., Yearim, A., Touboul, Y., and Ravid, S. (2012) The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration. Molecular biology of the cell 23, 591-601 31. Du, M., Wang, G., Ismail, T. M., Gross, S., Fernig, D. G., Barraclough, R., and Rudland, P. S. (2012) S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration. The Journal of biological chemistry 287, 15330-15344 32. Beach, J. R., Shao, L., Remmert, K., Li, D., Betzig, E., and Hammer, J. A., 3rd. (2014) Nonmuscle myosin II isoforms coassemble in living cells. Current biology : CB 24, 1160-1166 33. Shutova, M. S., Asokan, S. B., Talwar, S., Assoian, R. K., Bear, J. E., and Svitkina, T. M. (2017) Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility. The Journal of cell biology 216, 2877-2889 34. Shutova, M. S., Spessott, W. A., Giraudo, C. G., and Svitkina, T. (2014) Endogenous species of mammalian nonmuscle myosin IIA and IIB include activated monomers and heteropolymers. Current biology : CB 24, 1958-1968 35. Ayala-Sanmartin, J., Zibouche, M., Illien, F., Vincent, M., and Gallay, J. (2008) Insight into the location and dynamics of the annexin A2 N- terminal domain during Ca(2+)-induced membrane bridging. Biochimica et biophysica acta 1778, 472-482 36. Illien, F., Finet, S., Lambert, O., and Ayala-Sanmartin, J. (2010) Different molecular arrangements of the tetrameric annexin 2 modulate the size and dynamics of membrane aggregation. Biochimica et biophysica acta 1798, 1790-1796 37. Liu, L. (1999) Calcium-dependent self-association of annexin II: a possible implication in exocytosis. Cellular signalling 11, 317-324

8