RESUMEN
Diaphanous-related formins (DRFs) mediate GTPase-triggered actin rearrangements to regulate central cellular processes, such as cell motility and cytokinesis. The DRF FHOD1 interacts with the Rho-GTPase Rac1 and mediates formation of actin stress fibers in its deregulated form; the physiologically relevant activities and molecular mechanisms of endogenous FHOD1, however, are still unknown. Here we report that FHOD1 physically associates via the N-terminal part of its FH2 domain with the central domain of ROCK1. Although FHOD1 does not affect the kinase activity of ROCK1, the DRF is an efficient substrate for phosphorylation by ROCK1. Co-expression of FHOD1 and ROCK1 results in the generation of nonapoptotic plasma membrane (PM) blebs, to which the DRF is efficiently recruited. Blebbing induced by FHOD1 and ROCK1 depends on F-actin integrity, the Rho-ROCK cascade, and Src activity and is reminiscent of the recently described PM blebs triggered by expression of Src homology 4 (SH4) domain PM targeting signals. Consistently, endogenous FHOD1 is required in SH4 domain expressing cells for efficient PM blebbing and rounded cell morphology in two-dimensional cultures or three-dimensional matrices, respectively. Efficient association of FHOD1 with ROCK1, as well as recruitment of the DRF to blebs, depends on Src activity, suggesting that the functional interaction between both proteins is regulated by Src. These results define a role for endogenous FHOD1 in SH4 domain-induced blebbing and suggest that its activity is regulated by ROCK1 in a Src-dependent manner.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Proteínas Fetales/metabolismo , Proteínas Nucleares/metabolismo , Quinasas Asociadas a rho/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células CHO , Células COS , Estructuras de la Membrana Celular/genética , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas Fetales/genética , Forminas , Células HeLa , Humanos , Proteínas Nucleares/genética , Fibras de Estrés/genética , Fibras de Estrés/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho/genética , Dominios Homologos src/fisiología , Familia-src Quinasas/genéticaRESUMEN
A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.
Asunto(s)
Adenosilmetionina Descarboxilasa/biosíntesis , Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Leishmania donovani/enzimología , Ornitina Descarboxilasa/biosíntesis , Espermidina Sintasa/biosíntesis , Adenosilmetionina Descarboxilasa/genética , Animales , Butilaminas/farmacología , Desoxiadenosinas/farmacología , Resistencia a Medicamentos/genética , Eflornitina/farmacología , Leishmania donovani/genética , Ornitina Descarboxilasa/genética , Pruebas de Sensibilidad Parasitaria/métodos , Transducción de Señal/efectos de los fármacos , Espermidina Sintasa/genética , TransfecciónRESUMEN
Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.
Asunto(s)
Proteínas Fetales/fisiología , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Calorimetría , División Celular , Cromatografía , Cromatografía en Gel , Clonación Molecular , ADN/metabolismo , Proteínas Fetales/química , Forminas , Glutatión Transferasa/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Proteínas Nucleares/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Diaphanous-related formins (DRFs) are actin nucleators that mediate rearrangements of the actin cytoskeleton downstream of specific Rho GTPases. The DRF Formin Homology 2 Domain containing 1 (FHOD1) interacts with the Rac1 GTPase and induces the formation of and associates with bundled actin stress fibers. Here we report that active FHOD1 also coordinates microtubules with these actin stress fibers. Expression of a constitutive active FHOD1 variant in HeLa cells not only resulted in pronounced formation of FHOD1-actin fibers but also caused marked cell elongation and parallel alignment of microtubules without affecting cytokinesis of these cells. The analysis of deletions in the FH1 and FH2 functional regions revealed that the integrity of both domains was strictly required for FHOD1's effects on the cytoskeleton. Dominant-negative approaches demonstrated that filament coordination and cell elongation depended on the activity of the Rho-ROCK cascade, but did not involve Rac or Cdc42 activity. Experimental depolymerization of actin filaments or microtubules revealed that the formation of FHOD1-actin fibers was a prerequisite for the polarization of microtubules. However, only simultaneous disruption of both filament systems reversed the cell elongation induced by activated FHOD1. Thus, sustained cell elongation was a consequence of FHOD1-mediated actin-microtubule coordination. These results suggest filament coordination as a conserved function of mammalian DRFs.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Fetales/fisiología , Microtúbulos/metabolismo , Proteínas Nucleares/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Animales , Forma de la Célula/efectos de los fármacos , Citocalasina D/farmacología , Citocinesis/efectos de los fármacos , Proteínas Fetales/genética , Forminas , Células HeLa , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Mutación , Células 3T3 NIH , Nocodazol/farmacología , Proteínas Nucleares/genética , Transducción de Señal/fisiología , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Transfección , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The diaphanous-related formin homology 2 domain containing protein 1 (FHOD1) interacts with the Rac GTPase and activates the Rho-ROCK cascade leading to the formation of actin stress fibers. Here, we report the detection of homotypic interactions of FHOD1 in the yeast two-hybrid system, by co-immunoprecipitation and co-localization in mammalian cells. A predicted coiled-coil motif C-terminal to the core FH2 domain, but not the core FH2 domain itself, was critical for self-association of FHOD1. Deletion of both the coiled-coil motif and the core FH2 domain abrogated formation of actin stress fibers and activation of transcription of the serum response element by FHOD1. In contrast, these motifs were dispensable for the physical and functional interaction of FHOD1 with Rac1. Together, these results indicate that oligomerization of FHOD1 via the coiled-coil motif is a critical parameter for its biological activities.
Asunto(s)
Proteínas Fetales/química , Proteínas Fetales/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fibras de Estrés/ultraestructura , Activación Transcripcional , Secuencias de Aminoácidos/genética , Animales , Proteínas Fetales/genética , Forminas , Células HeLa , Humanos , Ratones , Modelos Moleculares , Células 3T3 NIH , Proteínas Nucleares/genética , Estructura Secundaria de Proteína/genética , Eliminación de Secuencia/genética , Fibras de Estrés/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.
Asunto(s)
Productos del Gen nef/fisiología , VIH-1/fisiología , VIH-1/patogenicidad , Microdominios de Membrana/enzimología , Microdominios de Membrana/virología , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Activación Enzimática , Productos del Gen nef/genética , VIH-1/genética , Humanos , Células Jurkat , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/fisiología , Linfocitos T/virología , Proteína de Unión al GTP cdc42/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Diaphanous related formins (DRFs) are part of the formin protein family that control morphogenesis, embryonic differentiation, cytokinesis, and cell polarity. DRFs organize the cytoskeleton in eukaryotic cells via the interaction with specific members of the Rho family of small GTPases including Rho, Rac, and Cdc42. This is best understood for Rho, which transmits signals to the actin cytoskeleton through the cooperation of its DRF effector mDia with ROCK (Rho-associated kinase). Here, we show that a constitutive active form of the Rac-interacting DRF FHOD1 (formin homology 2 domain containing 1) associates with F-actin in NIH3T3 cells, resulting in the formation of thick actin fibers. Cytoskeletal changes induced by FHOD1 correlated with the induction of serum response element transcription and were mediated by formin homology domains 1 and 2 of FHOD1. FHOD1-induced effects required the activity of the Rho-ROCK cascade that is targeted at a level downstream of Rho by the DRF. However, when the functional interaction of FHOD1 with individual GTPases was addressed, Rac but not Rho or Cdc42 bound to FHOD1 in cells and induced its recruitment to actin filaments and lamellipodia/membrane ruffles. Furthermore, activated FHOD1 interfered with lamellipodia formation. These results indicate that FHOD1 acts as an effector of Rac in actin rearrangements and transcriptional regulation and may provide a link for the Rac-dependent activation of the Rho cascade.
Asunto(s)
Actinas/metabolismo , Proteínas Fetales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Células 3T3 , Actinas/química , Animales , Western Blotting , Citoesqueleto/metabolismo , Forminas , GTP Fosfohidrolasas/química , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Seudópodos/química , Factores de Tiempo , Transcripción Genética , Transfección , Quinasas Asociadas a rhoRESUMEN
The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.