Cell migration, cytokinesis and the establishment and maintenance of cell morphology are fundamental force-requiring processes of all eukaryotic cells. Actin and myosin-II are essential cytoskeletal components of contractile processes in nonmuscle cells. Although it is recognized that myosin-II filament dynamics are under strict temporal and spatial control, the mechanisms controlling filament assembly in higher eukaryotes are not known. We are specifically addressing how covalent modification and noncovalent interactions with novel regulatory proteins mediate the subcellular localization, organization and assembly of myosin-II during chemotactic motility.
Phosphorylation of nonmuscle myosin-II on the heavy chain regulates filament assembly and is attributed to several kinases. Most recently, we showed that heavy chain phosphorylation regulates the chemotactic motility of tumor cells. Moreover, genes coding for proteins that modulate the myosin-II regulatory pathway are up-regulated in invasive tumor cells. Given these findings, we are examining the intermediary signaling pathways in tumor cells that regulate heavy chain phosphorylation and the subsequent effects on motility and invasion. We are using an interdisciplinary approach that combines biochemistry and structural biology to define the physical and chemical features underlying the regulation of myosin-II assembly by phosphorylation, and molecular and cellular techniques coupled with fluorescence microscopy to investigate how phosphorylation regulates myosin-II dynamics in vivo.
We are also studying S100A4, a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor metastasis and regulates tumor cell motility by promoting the monomeric, unassembled state of myosin-II. Thus S100A4 is an excellent target for investigating the mechanisms controlling the localized assembly/disassembly of myosin-II that are relevant to motility, development and metastasis. We are taking a global approach to dissecting S100A4 function; biochemical and structural approaches are being used to identify the mechanisms by which S100A4 regulates myosin-II assembly, intravital imaging studies will evaluate the impact of S100A4 expression on metastasis in live animal models; and a S100A4 knockout mouse has been developed to examine S100A4 function in normal physiology.
Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. We developed several assays to identify small molecules that disrupt the interaction of S100A4 with myosin-IIA. Our efforts are now focused on obtaining high-resolution x-ray structures of S100A4 bound to small molecule inhibitors to identify the chemical and structural determinants involved in S100A4 inhibition, and biochemical and cell-based analyses to evaluate the selectivity and potency of lead compounds. These studies will provide the biochemical and structural foundation for the design of second generation S100A4 inhibitors.
Representative Publications
Bresnick AR, Weber DJ, Zimmer DB (2015) S100 proteins in cancer. Nat Rev Cancer 15, 96-109.
Ramagopal U, Dulyaninova NG, Varney KM, Wilder PT, Nallamsetty S, Brenowitz M, Weber DJ, Almo SC, Bresnick AR (2013) Structure of the S100A4/myosin-IIA complex. BMC Struct Biol 13, 31.
Dulyaninova NG, Bresnick AR (2013) The heavy chain has its day: regulation of myosin-II assembly. Bioarchitecture 3, 77-85.
Ivkovic S, Beadle C, Noticewala S, Massey SC, Swanson KR, Toro LN, Bresnick AR, Canoll P and Rosenfeld SS (2012) Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens. Mol Biol Cell 23, 533-42.
Li Z-H, Dulyaninova NG, House RP, Almo SC and Bresnick AR (2010) S100A4 regulates macrophage chemotaxis. Mol Biol Cell 21, 2598-610.
Malashkevich V, Dulyaninova NG, Ramagopal UA, Liriano MA, Varney KM, Knight D, Brenowitz M, Weber DJ, Almo SC and Bresnick AR (2010) Phenothiazines inhibit S100A4 function by inducing protein oligomerization. PNAS 107, 8605-10.
Malashkevich V, Varney KM, Garrett SC, Wilder PT, Knight D, Charpentier TH, Ramagopal UA, Almo SC, Weber DJ and Bresnick AR (2008). Structure of Ca2+-bound S100A4 and its interaction with peptides derived from nonmuscle myosin-IIA. Biochemistry 47, 5111-26.
Garrett SC, Hodgson L, Rybin A, Toutchkine A, Hahn KM, Lawrence DS and Bresnick AR (2008) A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics. Biochemistry 47, 986-996.
Dulyaninova NG, House RP, Betapudi V and Bresnick AR (2007) Myosin-IIA heavy chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells. Mol Biol Cell 18, 3144-55.
Li Z-H and Bresnick AR (2006) The S100A4 metastasis factor regulates cellular motility via a direct interaction with myosin-IIA. Cancer Res 66, 5173-80.
Dulyaninova NG, Malashkevich V, Almo SC and Bresnick AR (2005) Regulation of myosin-IIA assembly and mts1 binding by heavy chain phosphorylation. Biochemistry 44, 6867-76.