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  • 1.
    Aase, Karin
    et al.
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ernkvist, Mira
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ebarasi, Lwaki
    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
    Jakobsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Majumdar, Arindam
    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
    Yi, Chunling
    Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
    Birot, Olivier
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ming, Yue
    Department of Clinical Neuroscience, Section of Ophthalmology and Vision, Karolinska Institutet, St Erik’s Hospital, SE-11284 Stockholm, Sweden.
    Kvanta, Anders
    Department of Clinical Neuroscience, Section of Ophthalmology and Vision, Karolinska Institutet, St Erik’s Hospital, SE-11284 Stockholm, Sweden.
    Edholm, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kissil, Joseph
    Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Shimono, Akihiko
    Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Chuou-ku, Kobe 650-0047, Japan.
    Holmgren, Lars
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Angiomotin regulates endothelial cell migration during embryonic angiogenesis2007In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 21, no 16, p. 2055-2068Article in journal (Refereed)
    Abstract [en]

    The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.

  • 2.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Activated Rho GTPases in Cancer-The Beginning of a New Paradigm2018In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 19, no 12, article id 3949Article, review/survey (Refereed)
    Abstract [en]

    Involvement of Rho GTPases in cancer has been a matter of debate since the identification of the first members of this branch of the Ras superfamily of small GTPases. The Rho GTPases were ascribed important roles in the cell, although these were restricted to regulation of cytoskeletal dynamics, cell morphogenesis, and cell locomotion, with initially no clear indications of direct involvement in cancer progression. This paradigm has been challenged by numerous observations that Rho-regulated pathways are often dysregulated in cancers. More recently, identification of point mutants in the Rho GTPases Rac1, RhoA, and Cdc42 in human tumors has finally given rise to a new paradigm, and we can now state with confidence that Rho GTPases serve as oncogenes in several human cancers. This article provides an expose of current knowledge of the roles of activated Rho GTPases in cancers.

  • 3.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Integration of signalling pathways regulated by small GTPases and calcium2004In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1742, no 1-3, p. 51-58Article, review/survey (Refereed)
    Abstract [en]

    The Ras superfamily of small GTPases constitutes a large group of structurally and functionally related proteins. They function as signalling switches in numerous signalling cascades in the cell. During the recent years, an increased awareness of a communication between signalling systems employing Ras-like GTPases and signalling systems employing calcium has emerged. For instance, the intensity of the activation of Ras-like GTPases is regulated by calcium-dependent mechanisms, acting on proteins that facilitate the activation or inactivation of the small GTPases. Other Ras-like GTPases have a direct influence on calcium signalling by regulating the activity of certain calcium channels. In addition, several small GTPases collaborate with calcium signalling in regulating cellular processes, such as cell adhesion, cell migration and exocytosis.

  • 4.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    The Intrinsic GDP/GTP Exchange Activities of Cdc42 and Rac1 Are A Critical Determinants for Their Specific Effects on Mobilization of the Actin Filament System2019In: CELLS, ISSN 2073-4409, Vol. 8, no 7, article id 759Article in journal (Refereed)
    Abstract [en]

    The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.

  • 5.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 298, no 2, p. 485-498Article in journal (Refereed)
    Abstract [en]

    The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor beta-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408-412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor beta-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.

  • 6.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The verprolin family of proteins: Regulators of cell morphogenesis and endocytosis2005In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, no 24, p. 5253-5259Article, review/survey (Refereed)
    Abstract [en]

    The verprolin family of proteins, WIP, CR16 and WIRE/WICH, has emerged as critical regulators of cytoskeletal organisation in vertebrate cells. The founding father of the family, verprolin, was originally identified in budding yeast and later shown to be needed for actin polymerisation during polarised growth and during endocytosis. The vertebrate verprolins regulate actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. Interestingly, also the vertebrate verprolins have been implicated in endocytosis, demonstrating that most of the functional modules in this fascinating group of proteins have been conserved from yeast to man.

  • 7.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The verprolins as regulators of actin dynamics.2006In: Actin-monomer-binding proteins., Austin, Texas: Landes Biosciences , 2006, p. 97-106Chapter in book (Refereed)
    Abstract [en]

    Verprolin is an actin-binding protein first identified in budding yeast Saccharomyces cerevisiae. The yeast verprolin is needed for actin polymerisation during polarised growth and during endocytosis. In vertebrate cells, three genes encoding Verprolin orthologues have been identified: WIP, CR16 and WIRE/WICH. The mammalian verprolins have been implicated in the regulation of actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. This review article will bring up to discussion the current understanding of the mechanisms underlying verprolin-dependent mobilisation of the actin filament system.

  • 8.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor2002In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 279, no 1, p. 21-33Article in journal (Refereed)
    Abstract [en]

    Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WIP-related). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRbeta cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckbeta. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.

  • 9.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Richnau, Ninna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pombe Cdc15 homology proteins: regulators of membrane dynamics and the actin cytoskeleton2006In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 31, no 12, p. 670-679Article, review/survey (Refereed)
    Abstract [en]

    Pombe Cdc15 homology (PCH) proteins have emerged in many species as important coordinators of signalling pathways that regulate actomyosin assembly and membrane dynamics. For example, the prototype PCH protein, Cdc15p of Schizosaccharomyces pombe, has a role in assembly of the contractile ring, which is needed to separate dividing cells. Recently, mammalian PCH proteins have been found to bind phospholipids and to participate in membrane deformation. These findings suggest that PCH proteins are crucial linkers of membrane dynamics and actin polymerization, for example, during the internalization of transmembrane receptors. Intriguingly, some members of the PCH protein family are mutated in neurodegenerative and inflammatory diseases, which has implications for the identification of cures for such disorders.

  • 10.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rho GTPases have diverse effects on the organization of the actin filament system2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 377, no Pt 2, p. 327-337Article in journal (Refereed)
    Abstract [en]

    The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.

  • 11.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Richnau, Ninna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Johansson, Ann-Sofi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 12, p. 2180-2194Article in journal (Refereed)
    Abstract [en]

    Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.

  • 12.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ruusala, Aino
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pacholsky, Dirk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Taking Rho GTPases to the next level: the cellular functions of atypical Rho GTPases2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 17, p. 3673-3679Article, review/survey (Refereed)
    Abstract [en]

    The Rho GTPases are influential regulators of signalling pathways that control vital cellular processes such as cytoskeletal dynamics, gene transcription, cell cycle progression and cell transformation. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, but this protein family actually harbours 20 members. Recently, the less known Rho GTPases have received increased attention. Many of the less studied Rho GTPases have structural, as well as, functional features which makes it pertinent to classify them as atypical Rho GTPases. This review article will focus on the critical aspects of the atypical Rho GTPases, RhoH, Wrch-1, Chp and RhoBTB. These proteins are involved in a broad spectre of biological processes, such as cytoskeletal dynamics, T-cell signalling and protein ubiquitinylation. We will also discuss the roles of atypical Rho GTPases as oncogenes or tumour suppressors, as well as their potential involvement in human diseases.

  • 13.
    Chiara, Federica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Goumans, Marie-José
    Forsberg, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Åhgrén, Aive
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rasola, Andrea
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellberg, Carina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 41, p. 42516-42527Article in journal (Refereed)
    Abstract [en]

    The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.

  • 14. Dib, Karim
    et al.
    Melander, Fredrik
    Axelsson, Lena
    Dagher, Marie-Claire
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Andersson, Tommy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 26, p. 24181-24188Article in journal (Refereed)
    Abstract [en]

    In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

  • 15.
    Edlund, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bu, Shizhong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Schuster, Norbert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Nils-Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    ten Dijke, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Transforming growth factor-beta1-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 32003In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 14, no 2, p. 529-544Article in journal (Refereed)
    Abstract [en]

    The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.

  • 16.
    Edlund, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Smad7 is required for TGF-ß-induced activation of the small GTPase Cdc422004In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 117, no Pt 9, p. 1835-1847Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-beta/Smad signaling cascade. In a previous study, we found that TGF-beta induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-beta signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-beta-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-beta-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-beta-dependent activation of Rho GTPases.

  • 17.
    Edlund, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Transforming growth factor-beta-induced mobilization of actin cytoskeleton required signaling by small GTPases Cdc42 and RhoA2002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 3, p. 902-914Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-beta receptors. We studied TGF-beta-induced rearrangements of the actin filament system and found that TGF-beta 1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-beta 1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-beta-induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.

  • 18.
    Edlund, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lee, So Young
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Grimsby, Susanne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Zhang, Shouthing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Landström, Maréne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Interaction between Smad7 and beta-catenin: importance for transforming growth factor beta-induced apoptosis2005In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 4, p. 1475-1488Article in journal (Refereed)
    Abstract [en]

    Members of the transforming growth factor beta (TGF-beta) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with beta-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-beta-dependent manner. Smad7 was found to be required for TGF-beta1-induced accumulation of beta-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-beta-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3beta phosphorylated on Ser9 was prevented, as well as the TGF-beta-induced association between beta-catenin and LEF1. Notably, the observed physical association of Smad7 and beta-catenin was found to be important for TGF-beta-induced apoptosis, since suppression of beta-catenin expression by small interfering RNA decreased the apoptotic response to TGF-beta.

  • 19.
    Gizatullina, Zemfira Z.
    et al.
    Ludwiginstitutet för Cancerforskning.
    Grapengiesser, Eva
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Shabalina, Irina G.
    Nedergaard, Jan
    Heldin, Carl-Henrik
    Ludwiginstitutet för Cancerforskning.
    Aspenström, Pontus
    Ludwiginstitutet för Cancerforskning.
    Effect of transforming growth factor-beta on calcium homeostasis inprostate carcinoma cells.2003In: Biochem Biophys Res Commun, Vol. 304, p. 643-Article in journal (Refereed)
    Abstract [en]

    Ca(2+) plays a fundamental role in the control of a variety of cellular functions, in particular, in energy metabolism and apoptosis. In this study, we show that TGF-beta at concentrations of 0.1-1.0 ng/ml transiently increases the level of intracellular Ca(2+) ([Ca(2+)](in)) in human prostate carcinoma, PC-3U, cells. Experiments with mitochondrial inhibitors (oligomycin and antimycin A) and an inhibitor of endoplasmic reticulum Ca(2+) uptake (BHQ) implied that the effect of TGF-beta1 was due to an effect on the mitochondria. TGF-beta1 treatment resulted in a decrease in ATP synthesis and to a depolarisation, leading to a release of Ca(2+) from mitochondria and decreased activity of the Ca(2+) pumps. Analysis of the mitochondria within the PC-3U cells by polarography and membrane potential-sensitive dye (Rhodamine 123) confirmed that under these experimental conditions, TGF-beta1 inhibited ATP synthesis and depolarised the mitochondria. The results implicate that TGF-beta1 affects the function of the mitochondria and may be of significance for the understanding of the proapoptotic effect of TGF-beta1 in these cells.

  • 20. Jaffe, Aron B
    et al.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hall, Alan
    Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 4, p. 1736-1746Article in journal (Refereed)
    Abstract [en]

    Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.

  • 21. Lundström, Annika
    et al.
    Gallio, Marco
    Englund, Camilla
    Steneberg, Pär
    Hemphälä, Johanna
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Keleman, Krystyna
    Falileeva, Ludmilla
    Dickson, Barry J
    Samakovlis, Christos
    Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons2004In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 18, no 17, p. 2161-2171Article in journal (Refereed)
    Abstract [en]

    Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.

  • 22.
    Richnau, Ninna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Farsad, Khashayar
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 320, no 3, p. 1034-1042Article in journal (Refereed)
    Abstract [en]

    RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events.

  • 23.
    Ruusala, Aino
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Isolation and characterisation of DOCK8, a member of the DOCK180-related regulators of cell morphology2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 572, no 1-3, p. 159-166Article in journal (Refereed)
    Abstract [en]

    In a yeast two-hybrid system screen for Cdc42-interacting proteins, we identified a protein with similarity to the CrkII-binding protein DOCK180. A cDNA clone of this protein, designated DOCK8, encoded a gene-product of 1701 amino acid residues with a molecular mass of 190 kDa. Immunofluorescence staining showed that transiently transfected HA-tagged DOCK8, as well as endogenous DOCK8, was present at the cell edges in areas undergoing lamellipodia formation. Transient transfection of a C-terminal fragment of DOCK8 resulted in the formation of vesicular structures. Interestingly, these vesicles also contained filamentous actin. These data suggest an involvement of DOCK8 in processes that affect the organisation of filamentous actin.

  • 24.
    Ruusala, Aino
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 5, p. 1802-14Article in journal (Refereed)
    Abstract [en]

    The Cdc42-like GTPase Wnt responsive Cdc42 homolog 1 (Wrch1) has several atypical features; it has an N-terminal proline-rich extension that confers binding to SH3 domains, and it harbors an extremely high intrinsic nucleotide exchange activity, which overrides the normal GTPase activity. As a result, Wrch1 resides mainly in the active, GTP-loaded conformation under normal cellular conditions. We have previously shown that ectopic expression of Wrch1 in fibroblasts resulted in an altered cell morphology visible as a formation of filopodia, a loss of stress fibers, and a reduction in focal adhesions. Here, we show that Wrch1 binds to the nonreceptor tyrosine kinase Pyk2. The interaction required Wrch1 to be in a GTP conformation and also required an intact N-terminal proline-rich extension as well as an intact effector loop. Wrch1 requires Pyk2 in imposing the cytoskeletal effects, seen as the formation of filopodia, since treatment of cells with a Pyk2-specific small interfering RNA abrogated this response. Interestingly, we found that the presence and activity of Src were needed for the formation of a Wrch1-Pyk2 complex as well as for the Wrch1-induced formation of filopodia. We propose a model in which Pyk2 and Src function to coordinate the Wrch1-dependent effects on cytoskeletal dynamics.

  • 25.
    Saras, Jan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Wollberg, Patrick
    Aspenström, Pontus
    Ludwiginstitutet för Cancerforskning.
    Wrch1 is a GTPase-deficient Cdc42-like protein with unusual binding characteristics and cellular effects.2004In: Exp Cell Res, ISSN 0014-4827, Vol. 299, no 2, p. 356-69Article in journal (Refereed)
    Abstract [en]

    The Rho family of small GTPases controls many biological processes, including cytoskeletal regulation, membrane trafficking, cell adhesion, cell polarization, transcriptional activity, apoptosis, and cell proliferation. Wrch1, which belongs to the Cdc42 subfamily, is one of the least characterized family member. Despite its homology to other Cdc42-like proteins, we found that Wrch1 has unique characteristics. Biochemical experiments showed that Wrch1 has no detectable GTPase activity in vitro and that its intrinsic nucleotide exchange rate is very high in comparison to Cdc42. Furthermore, NIH3T3 cells transiently transfected with Wrch1 showed an up-rounded, retracted phenotype. In addition, Wrch1 was shown to be more efficient than Cdc42 in triggering the formation of filopodia. Serum stimulation of cells expressing Wrch1 induces vigorous membrane blebbing, a phenomenon dependent on the activity of ROCK. In a search for proteins interacting with Wrch1, PAK1 and NCKbeta were identified as binding partners. Interestingly, the interaction to NCKbeta was shown to be mediated via PxxP motifs present in an N-terminal extension of Wrch1 to the second and third SH3 domains of NCKbeta.

  • 26. Westerberg, Lisa
    et al.
    Greicius, Gediminas
    Snapper, Scott B.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Severinson, Eva
    Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes.2001In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 98, no 4, p. 1086-1094Article in journal (Refereed)
    Abstract [en]

    Patients with the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) have lymphocytes with aberrant microvilli, and their T cells, macrophages, and dendritic cells are impaired in cytoskeletal-dependent processes. WAS is caused by a defective or a missing WAS protein (WASP). Signal mediators interleukin-4 (IL-4) and CD40 are important for actin-dependent morphology changes in B cells. A possible function of WASP and its interacting partners, Cdc42 and Rac1, was investigated for these changes. It was found that active Cdc42 and Rac1 induced filopodia and lamellipodia, respectively, in activated B cells. Evidence is given that IL-4 has a specific role in the regulated cycling of Cdc42 because IL-4 partially and transiently depleted active Cdc42 from detergent extract of activated B cells. WASP-deficient B lymphocytes were impaired in IL-4-- and CD40-dependent induction of polarized and spread cells. Microvilli were expressed on WASP-deficient B cells, but they appeared shorter and less dense in cell contacts than in wild-type cells. In conclusion, evidence is provided for the involvement of Cdc42, Rac1, and WASP in the cytoskeletal regulation of B lymphocytes. Aberrations in WASP-deficient B lymphocytes, described here, provide further evidence that WAS is a cytoskeletal disease of hematopoietic cells.

1 - 26 of 26
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