<p>The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates <i>in vivo</i>, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses.</p><p>In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42</p><p>The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.</p>

The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates in vivo, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses. In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42 The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.