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11 months ago

2005 Scientific Report

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  • Signaling
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Signal transduction and

Signal transduction and spatially controlled assembly of F-actin networks One well-characterized actin nucleator, the Arp2/3 complex, induces the formation of branched actin filaments. Arp2/3 works by complexing with G-actin or by binding to the side of preexisting filaments. Its nucleation and filament-binding activity is tightly regulated by interactions with nucleation-promoting factors (NPFs), the most prominent being the WASp/Scar family. WASp is an autoregulated molecular switch controlled by yet other switch-like proteins such as Cdc42, a Rho family small GTPase. Thus, NPFs integrate signals controlling growth factor–stimulated actin nucleation and branching. Cdc42-activated WASp induction of Arp2/3 activity is shown schematically in Fig. 1A. The mammalian Diaphanous-related formins Formins are a highly conserved family of proteins implicated in a diverse array of cellular functions including the cytoskeletal remodeling events necessary for cytokinesis, bud formation in yeast, establishment of cell/organelle polarity, and endocytosis. Formins have the ability to stabilize microtubules, which (like F-actin) are assembled by tightly controlled cycles of polymerization and depolymerization. The mammalian Diaphanous-related formin (mDia) proteins are a subfamily of formins that share a loosely conserved Rho GTPase-binding domain (GBD) in the amino terminus and a highly conserved Diaphanous-autoregulatory domain (DAD) in the carboxy terminus. The GBDs can interact with their internal DAD partners in vitro, leading to the autoregulation model depicted in Fig. 1B. The model shows that while the formin proteins dimerize through their FH2 domains, it is the GBD-DAD interaction that is the linchpin of autoregulation. GTP-bound Rho can interact with the GBD and interrupt the autoinhibited conformation, leading to nucleation and elongation of nonbranched actin filaments. It has been unclear whether GTPase binding simply activates the mDia proteins’ ability to nucleate actin, or provides other signals that direct subcellular targeting and recruitment of mDia-associated proteins. Another question is, do mDia proteins activated in specific cellular contexts (i.e., on vesicles or at sites of adhesion) associate with or work in parallel with other modifiers of actin polymerization to generate site-specific F-actin networks? We are studying such questions using FRET technology. Site-specific interactions between Rho GTPases and mDia proteins Fluorescence resonance energy transfer (FRET) is a powerful technique that allows us to assay protein-protein interactions in cells by using two fluorophores, in this case, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). When fused to the GTPase and excited at the appropriate wavelength, CFP acts as a fluorescent donor which then excites YFP, which is fused to a Drf protein (Fig. 1B). FRET Figure 1. Drfs are actin nucleators whose activity is regulated through interactions with small GTPases. A) A model for formin (mDia1-3) and WASp collaborating in cells with activated Cdc42, in which Cdc42 interacts with mDia2 in cells at specific sites associated with membrane protrusions. In this model, activated WASp nucleates branched filaments from the side of mDia2 nucleated “mother” filaments; alternatively, mDia2 binds to and processively elongates filaments after nucleation by Arp2/3. B) GTP-bound Rho GTPase binding disrupts intramolecular interactions between the GBD and DAD of a DRF. If the GTPase and GBD are linked to fluorophores (ECFP and EYFP), the proximity of the two can be determined by FRET. 10

occurs only when the donor/acceptor pair is in close proximity (less than 30 Å) . Fusion proteins are expressed following microinjection of their expression plasmids into cells, which are then fixed 4 h later. We have shown that YFP-mDia2 is expressed with CFP- Cdc42, primarily at the leading cell edge (or cortex) and at the microtubule-organizing center. In other experiments, we have shown that this interaction depends upon the integrity of the CRIB motif within the mDia2 GBD. CRIB motifs are necessary for binding to the GTPase. Our observations indicate that one particular GTPase-formin pair, Cdc42 and mDia2, may have a role in remodeling actin at the cell edge. What this pair contributes to actin or microtubule dynamics at the MTOC, however, remains an open question. We speculate that the pair may participate in microtubule regulation at the minus (–) end of the tubules, which (like actin) are assembled and disassembled in a polarized fashion. Other GTPase-formin pairs may be working at the plus end to direct them to focal adhesions or other sites. From collaborative efforts with the Gundersen lab, it has been shown that mDia1 and mDia2 can complex with the MT(+)-end binding proteins APC and EB1. In contrast to the speculative role of Cdc42 and mDia2, RhoB is known to have a role in endocytic or vesicular trafficking, and it interacts with mDia2 on endosomes (Fig. 2). This result is consistent with our discovery of both mDia1 and mDia2 on endosomes. The expression of either activated RhoB or deregulated versions of mDia1, mDia2, and mDia3 blocks the movement of vesicles and increases their number. One interpretation is that expression of RhoB or deregulated mDia1–3 triggers an inappropriate transition from fast microtubule-dependent transport to actin-dependent transport. Formins as anti-cancer drug targets A dynamic cytoskeleton is required for tumor cell growth. Drugs that stabilize the cytoskeleton are emerging as effective anti-cancer therapeutics. For example, Taxol binds directly to the components that comprise the microtubule cytoskeleton and blocks their dynamics. A cyclopeptide derived from a sea sponge, jasplakinolide, has similar effects on actin. Figure 2. RhoB interacts with mDia2 on vesicles. CFP-fused activated RhoB-G14V and YFP-fused mDia2 were co-injected into cells and FRET was assessed 4 h later. A FRET signal is observed between activated RhoB and mDia2 upon endosomes. These data indicate that individual GTPase-formin pairs would appear to be functionally distinct, promoting the formation of different actin structures at different sites. Since both RhoB and Cdc42 have been implicated in endocytosis and vesicle trafficking, it is possible that both GTPases use mDia2 sequentially or in parallel as effectors to transport cargo within cells. Previously, we found that a peptide derived from the DAD region of mDia proteins, when expressed in cells, stabilized both the actin and microtubule cytoskeletons. Because the mechanism of DAD action is unique—it binds to cellular formins and disrupts their normal autoregulatory mechanism—DAD represents a novel class of anti-tumor drugs. Because DAD is unable to enter cells as a drug, we have begun searching for functional analogs of DAD that could have similar properties under a drug-development program funded by the National Cancer Institute. We hypothesize that by deregulating specific mDia molecules in tumor cells, we can arrest dynamic remodeling of the cytoskeleton, a process required for cell motility and cytokinesis. We will characterize the structural and functional requirements for DADinduced cell death and develop a high-throughput screen for the identification of novel molecules that can deregulate mDia proteins and kill tumor cells. Our objectives are to initiate a drug discovery program by validating mDia proteins as molecular targets in cancer and to determine the physical requirements for DAD interactions with the mDia GBD. 11

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