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Biomedical subjects

M F Olson

Publications and source records attributed to M F Olson.

At least 19 recordsLinked to original sources
PubMed2006

Stability of p21Waf1/Cip1 CDK inhibitor protein is responsive to RhoA-mediated regulation of the actin cytoskeleton.

The proto-oncogene Ras GTPase stimulates transcription of p21Waf1/Cip1 (p21), which is repressed by the RhoA GTPase. We previously showed that Ras also elevates p21 protein levels by reducing its proteasome-mediated degradation. Therefore, we investigated whether RhoA also influenced p21 protein degradation. Pulse-chase analysis of p21 protein stability revealed that inhibitors of Rho function, which disrupt filamentous actin (F-actin), drastically slowed p21 degradation. Direct F-actin disruption mimicked Rho inhibition to stabilize p21. We found that Rho inhibition, or F-actin disruption, activated the JNK stress-activated protein kinase, and that interfering with JNK signalling, but not p38, abrogated p21 stabilization by Rho inhibition or F-actin-disrupting drugs. In addition, Ras-transformation led to increased constitutive JNK activity that contributed to the elevated p21 protein levels. These data suggest that p21 stability is influenced by a mechanism that monitors F-actin downstream of Rho, and which acts through a pathway involving activation of JNK. These results may have significant implications for therapies that target Rho-signalling pathways to induce p21-mediated cell-cycle arrest.

Actins↗
PubMed2002

Rho GTPase signalling pathways in the morphological changes associated with apoptosis.

The killing and removal of superfluous cells is an important step during embryonic development, tissue homeostasis, wound repair and the resolution of inflammation. A specific sequence of biochemical events leads to a form of cell death termed apoptosis, and ultimately to the disassembly of the dead cell for phagocytosis. Dynamic rearrangements of the actin cytoskeleton are central to the morphological changes observed both in apoptosis and phagocytosis. Recent research has highlighted the importance of Rho GTPase signalling pathways to these changes in cellular architecture. In this review, we will discuss how these signal transduction pathways affect the structure of the actin cytoskeleton and allow for the efficient clearance of apoptotic cells.

Actin Cytoskeleton↗
PubMed2001

Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility.

Transformation by oncogenic Ras requires the function of the Rho family GTPases. We find that Ras-transformed cells have elevated levels of RhoA-GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho-GTP are not a direct consequence of Ras signalling but are selected for in response to sustained ERK-MAP kinase signalling. While the elevated levels of Rho-GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibres in transformed cells. We show that the sustained ERK-MAP kinase signalling resulting from transformation by oncogenic Ras down-regulates ROCK1 and Rho-kinase, two Rho effectors required for actin stress fibre formation. The repression of Rho- dependent stress fibre formation by ERK-MAP kinase signalling contributes to the increased motility of Ras-transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibres and inhibits the motility of Ras-transformed fibroblasts. We propose a model in which Ras and Rho signalling pathways cross-talk to promote signalling pathways favouring transformation.

Animals↗
PubMed2001

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I.

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

3T3 Cells↗
PubMed2000

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain.

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Ras protein signalling.

Ras proteins were identified through their association with cell transformation. Since then they have been shown to regulate cell growth, differentiation and apoptosis, as well as influencing processes such as cell migration and neuronal activity. Ras regulates a number of signalling molecules by translocating them to the plasma membrane for activation. An emerging concept is that Ras acts as a branchpoint in signal transduction because it orchestrates the activity of multiple signalling pathways to regulate diverse cellular functions. This implies a degree of selectivity in the ability of Ras to activate particular arms of each pathway, but the mechanisms by which this is achieved are not known. Ras is also an important regulator of immune function and in this review, we summarise current understanding of Ras regulation and function and discuss some new aspects of Ras signalling where understanding is less clear.

Animals↗
PubMed1998

Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1.

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.

3T3 Cells↗
PubMed1998

Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 is thought to be Ras independent. Nevertheless, stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras blocked activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC was not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases.

Animals↗
PubMed1997

Distinct roles for DH and PH domains in the Lbc oncogene.

Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which specifically acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent fibroblasts and Rho-dependent actin stress fiber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress fibers and for efficient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.

3T3 Cells↗
PubMed1997

Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein.

Ras proteins act as molecular switches, responding to signals by entering the active GTP-bound, rather than the inactive GDP-bound, state. The inhibition of normal Ras proteins by microinjection of neutralizing antibody or expression of dominant-negative mutants has shown that Ras signalling is required for growth factors to stimulate DNA synthesis [1] [2], but the link between Ras and the cell-cycle machinery is not clear. Regulation of the phosphorylation state of the retinoblastoma protein (pRb), the product of the tumour suppressor gene Rb, is a key event in the progression of cells from G1 phase into S phase. In growth-arrested or early G1 cells, pRb is hypophosphorylated and binds to transcription factors of the E2F family [3]. These pRb-E2F complexes act to suppress gene transcription required for entry into DNA synthesis either by preventing E2F from stimulating transcription or by actively repressing transcription [4]. During G1, cyclin-dependent kinases (CDKs) become activated and phosphorylate pRb at multiple sites, leading to the dissolution of pRb-E2F complexes and gene transcription [5]. Here, we have tested the hypothesis that Ras signalling is required for the inactivation of pRb. A neutralizing antibody directed against p21Ras was microinjected into cells derived from mutant mouse embryos that lack Rb or CDK inhibitors (CDKIs). Cells without pRb or the p16 CDKI were more resistant to the inhibitory effects of the anti-Ras antibody. DNA synthesis in some tumour cell lines was completely resistant to the anti-Ras injection, indicating that p21Ras is required for pRb inactivation but also has other functions in cell-cycle progression.

Animals↗
PubMed1996

Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases.

BACKGROUND: Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation. RESULTS: Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. CONCLUSIONS: We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

3T3 Cells↗
PubMed1996

Guanine nucleotide exchange factors for the Rho GTPases: a role in human disease?

The Rho family of low molecular weight GTP-binding proteins control important aspects of cell shape, adhesion, movement and growth. The DBL-homology (DH) protein family of upstream regulators of Rho GTPases has recently been identified, and deregulated expression of these proteins can have dramatic cellular consequences. This review examines the possible role of DH proteins and Rho GTPases in oncogenesis, metastasis and development.

Animals↗
PubMed1995

An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1.

Members of the Rho family of small guanosine triphosphatases (GTPases) regulate the organization of the actin cytoskeleton; Rho controls the assembly of actin stress fibers and focal adhesion complexes, Rac regulates actin filament accumulation at the plasma membrane to produce lamellipodia and membrane ruffles, and Cdc42 stimulates the formation of filopodia. When microinjected into quiescent fibroblasts, Rho, Rac, and Cdc42 stimulated cell cycle progression through G1 and subsequent DNA synthesis. Furthermore, microinjection of dominant negative forms of Rac and Cdc42 or of the Rho inhibitor C3 transferase blocked serum-induced DNA synthesis. Unlike Ras, none of the Rho GTPases activated the mitogen-activated protein kinase (MAPK) cascade that contains the protein kinases c-Raf1, MEK (MAPK or ERK kinase), and ERK (extracellular signal-regulated kinase). Instead, Rac and Cdc42, but not Rho, stimulated a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun NH2-terminal kinase or stress-activated protein kinase). Rho, Rac, and Cdc42 control signal transduction pathways that are essential for cell growth.

3T3 Cells↗
PubMed1995

Direct involvement of the small GTP-binding protein Rho in lbc oncogene function.

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco-lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

3T3 Cells↗
PubMed1994

Receptor-G-protein coupling in desensitization-resistant mutant adrenal cells.

Mutant isolates from the Y1 mouse adrenocortical tumor cell line (designated Y1DR) were previously shown to resist homologous desensitization of adenylyl cyclase via endogenous ACTH receptors or transfected beta 2-adrenergic receptors. To further localize the site of the DR mutation, we examined the agonist-binding properties of the transfected beta 2-adrenergic receptor in parental Y1 cells and the DR mutant before and after desensitization with the beta-adrenergic agonist isoproterenol. Binding studies were carried out using [125I]iodocyanopindolol as the labeled ligand and isoproterenol as the competing agonist. We found that the DR mutant has a greater number of high affinity receptors in the basal state than does the Y1 parent (70-80% vs. < or = 50%) and that treatment of membranes from parent or mutant cells with guanyl-5'-yl imidodiphosphate and NaCl converts all of the high affinity sites to low affinity sites. After desensitization with isoproterenol, only low affinity binding sites are detected in parental Y1 cells, whereas the DR mutants retain an appreciable number of high affinity receptors. These results indicate that the DR mutant may resist desensitization by affecting agonist-induced uncoupling of receptors from their guanyl nucleotide-binding regulatory G-proteins.

Adrenal Glands↗
PubMed1993

Molecular basis for the 3',5'-cyclic adenosine monophosphate resistance of Kin mutant Y1 adrenocortical tumor cells.

A series of mutant cell lines (Kin) were previously isolated from Y1 adrenocortical tumor cells based on their ability to resist the growth-inhibitory effects of 8-bromo cAMP. In these Kin clones, cAMP-dependent protein kinase (cAMPdPK) was resistant to activation by cAMP as the consequence of mutations affecting the type I regulatory subunit (RI) of the enzyme. This study shows that the cAMP-resistant phenotypes of mutant clones Kin-2, Kin-7, and Kin-8 were associated with single base changes causing substitutions, respectively, of Glu for Gly200, Trp for Arg334, and Asp for Gly324 in the RI protein. By expressing the mutant Trp334 and Asp324 forms of RI under the control of an inducible promoter in Y1 cells, the causal relationship between these RI mutations and impairment of cAMP-stimulated adrenocortical responses was studied. Expression of the mutant RI forms rendered cAMPdPK resistant to activation by cAMP and decreased cAMP-stimulated cell rounding, steroid production, and growth inhibition. These observations indicate that the cAMP-resistant phenotype of Kin mutant clones resulted specifically from single mutational events in RI and thus establish the importance of cAMPdPK as an essential regulator of adrenocortical function. Unlike the original Kin mutant clones, transformants expressing the mutant forms of RI had adenylyl cyclases that were resistant to activation by ACTH, forskolin, or sodium fluoride. These results indicate that there may be a hitherto unappreciated mechanism of regulation of adenylyl cyclase activity by cAMPdPK.

8-Bromo Cyclic Adenosine Monophosphate↗