Post-Translational Regulation of the Mammalian Formin Daam1 Senior Thesis Presented to The Faculty of the School of Arts and Sciences Brandeis University Undergraduate Program in Biology Bruce Goode, Advisor In partial fulfillment of the requirements for the degree of Bachelor of Science By Jessica Kirshner May 2015 Copyright by Jessica Kirshner Committee members: Name: Bruce Goode Signature: . Name: Avital Rodal Signature: . Name: Joan Press Signature: . Abstract: Daam1 is a mammalian formin that, like other formins, has a catalytic FH2 domain. However, unlike other formins, the linker region differs in that it contains a small antiparallel beta sheet structure that partially occludes the two actin binding sites of the FH2 domain. This feature makes unmodified Daam1 a poor actin nucleator in vitro, as compared to most other formins. Here, we use total internal reflection fluorescence (TIRF) microscopy and bulk fluorescence assembly assays to demonstrate that Src kinase can phosphorylate Daam1 in vitro to increase its nucleation activity. This effect is dampened in a point mutant of Daam1 lacking the critical tyrosine at residue 652. We propose that phosphorylation by Src kinase in vivo serves as a regulatory mechanism in controlling Daam1 function by releasing the protein from its novel autoinhibited conformation to stimulate actin assembly. Experiments in NIH3T3 cells support this hypothesis by demonstrating that inhibiting Src family kinases reduce the number of filopodia protruding from the cells. The biological importance of this mechanism will continue to be elucidated using in vivo experiments on Daam1 mutants in live cells. Introduction: Eukaryotic cells depend upon actin to maintain their integrity and perform vital cellular functions so much so that this protein is present in all types of eukaryotic cells; the only known exception to this observation being the sperm of nematodes1. Its biological importance causes is to be one of the most abundant proteins on earth and in cells, with a cellular concentration of 100 µM2. With such high cellular concentrations, it is unsurprising that it fulfills a host of functions, which include providing tracks for movement of intracellular materials, providing mechanical support to maintain cell shape, providing force to aid in cell motility, and aiding in vesicle internalization2. The ability to perform these functions relies on the spontaneous conversion of monomeric G-actin to filamentous F-actin at physiological conditions to produce stable, polar filaments2, 3. This process occurs in four steps: monomer activation, nucleation, elongation, and finally, annealing4. Monomer activation refers to a fast step in which the divalent cation, magnesium, binds to actin monomers, thus inducing a change in conformation that makes them capable of polymerizing5. Nucleation describes the rate-limiting initial assembly of actin monomers into unstable dimers and trimers. Once, however, a fourth monomer is added to a trimer, the nucleus is formed, and is more likely to grow into a filament than to dissociate back to monomeric actin4. Nucleus formation allows for filament elongation that requires the coupled hydrolysis of ATP. Because all actin monomers are polymerized in the same orientation, actin filaments are polar and have separate rate constants describing association and dissociation from each end. The barbed end of the filament allows more rapid actin assembly and slower disassembly, while the pointed end demonstrates slower monomer addition, but faster disassembly4. Annealing is the process that describes end-to-end joining of actin filaments4. Due to the spontaneous nature of the actin polymerization process, many accessory proteins exist in the cell to regulate both actin assembly and disassembly and prevent deleterious outcomes for the cell. Over 100 proteins, of various functions, play this central role in eukaryotic cells2. These proteins participate in polymerization initiation, length control of the filament, assembly regulation, actin turnover regulation, cross-linking filaments, severing filaments, initiating branch formation, and others2. Many of these associated proteins function to inhibit spontaneous polymerization so that the cell may maintain a regulated environment. This necessitates other associated proteins that use active mechanisms to catalyze actin nucleation and elongation while preventing disassembly, in the process. The formin family of proteins is responsible for this task. Formins are integral cellular components that are conserved in plants, fungi, and animals6. Mammals express 15 different formins that all regulate cytoskeletal dynamics. Though formins play a role in regulating microtubule dynamics as well, we focus on their function in directing the assembly of linear actin filaments. Unlike other proteins that catalyze actin polymerization, formins are thought to be unique in their ability to catalyze both nucleation and elongation of the filament6. They directly aid actin assembly for processes such as morphogenesis, adhesion, division, and motility. The importance of formins in these processes make them a target of research because not only will their understanding advance our comprehension of the fundamentals of basic biology, but they have been implicated in a range of diseases, most notably, cancer, due to their role in cell division and motility. Increased expression of the formin FMNL1 is associated with leukemia7 and non-Hodgkin’s lymphoma8. Increased expression of other formins have been linked to metastasis and progressive invasion of cancers9,10. One case reports a congenital heart defect in a patient who has a single copy deletion of the Daam1 gene, which codes for the mammalian formin, Daam111. These data, along with various other studies linking formins and their associated proteins to cancer, immunological problems, cardiac problems, drive us to study them in great depth. As a class, formins are large proteins that exist as homodimers and have multiple domains, allowing them to interact with many binding partners to perform their function6. The amino terminus of the protein serves regulatory roles while the carboxy terminus is more influential on the catalytic activity of the protein6. The N-terminal regulatory region consists of a Rho-binding domain (RBD), a Dia inhibitory domain (DID), a dimerization domain, and a coiled coil (CC) domain. The C-terminus of the protein is comprised of the proline rich formin homology 1 (FH1) domain and the formin homology 2 (FH2) domain. Additionally, there is a DAD domain C-terminal to the FH2 domain that plays an auto- inhibitory role in formin activity by binding to the N-terminal DID domain6. The FH1 domain structure seems to lack an organized structure and is therefore unknown as of yet6. Its function is to bind profilin-bound actin monomers to accelerate elongation12. Profilin is a ubiquitously expressed protein that binds both actin monomers and the polyproline tract of the FH1 domain15. Most G-actin exists in a complex with profilin, and therefore, assembly is dependent upon the ability to catalyze polymerization using profilin-actin as a substrate14. The FH2 domain is well characterized, as several crystal structures exist for this domain. The FH1 and FH2 domains are responsible for the formin’s ability to nucleate and elongate actin, though the FH2 domain alone is sufficient to catalyze actin nucleation, in vitro13. Because this domain lacks the ability to bind actin monomers,13 it is believed that it is stabilizing the otherwise unstable spontaneously formed dimers and trimers to catalyze nucleation13. Presence of the FH1 domain significantly increases the catalytic ability of the formin13 by ferrying profilin-actin monomers to the FH2 domain22. Once a seed is formed, the two halves of the FH2 dimer alternate contact with the filament, moving in a step-wise fashion along the growing barbed end of the filament, incorporating a new subunit with each step while preventing capping of the filament by capping proteins16,28. The rate of this monomer addition has been quantified to be as high as 100 subunits per second6. Despite the motor nature of formins in “walking” along the barbed end of a filament, it is important to note that formins do not rely on the hydrolysis of ATP, or other nucleotides to supply energy for such movement. Recent studies suggest that the movement of a formin is coupled to a release in free energy associated with the polymerization of actin, thus allowing for its movement17. Though all formins function in mechanistically similar manners, and share general domain structures, we focus on one particular mammalian formin, Daam1. Daam1 (disheveled associated activator of morphogenesis) is a ubiquitously expressed formin with particular importance in heart morphogenesis18, gastrulation19, neuron development20, and filopodia formation20. Like other formins, Daam1 has a catalytic FH2 domain, however, the linker region of this formin differs from others in that it contains a small additional antiparallel beta sheet structure that partially occludes the two actin binding sites of the FH2 domain21. This feature makes unmodified Daam1 a poor actin nucleator in vitro, as compared to most other formins. Our understanding of Daam1’s structure comes, principally, from the crystal structure of a large C-terminal fragment of the formin, solved by Lu, et al. in 200721. The crystal structure revealed five sub-domains that comprise the FH2 domain, namely: the lasso, a linker, a globular knob