RESUMO
BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Células 3T3 , Actinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Células HeLa , Humanos , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.
Assuntos
Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Transporte Biológico , Complexo Dinactina , proteínas de unión al GTP Rab7RESUMO
Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance [1-3]. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance [4-7]. Using yeast two-hybrid screening and in vitro interaction assays, we show that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induced similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. Our findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Drosophila , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genéticaRESUMO
MHC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments that are generated in the endosomal pathway. The class II molecules are targetted to early lysosomal structures, termed MIIC, where they interact with antigenic fragments and are subsequently transported to the cell surface. We previously visualised vesicular transport of MHC class II-containing early lysosomes from the microtubule organising centre (MTOC) region towards the cell surface in living cells. Here we show that the MIIC move bidirectionally in a 'stop-and-go' fashion. Overexpression of a motor head-deleted kinesin inhibited MIIC motility, showing that kinesin is the motor that drives its plus end transport towards the cell periphery. Cytoplasmic dynein mediates the return of vesicles to the MTOC area and effectively retains the vesicles at this location, as assessed by inactivation of dynein by overexpression of dynamitin. Our data suggest a retention mechanism that determines the perinuclear accumulation of MIIC, which is the result of dynein activity being superior over kinesin activity. The bidirectional nature of MIIC movement is the result of both kinesin and dynein acting reciprocally on the MIIC during its transport. The motors may be the ultimate targets of regulatory kinases since the protein kinase inhibitor staurosporine induces a massive release of lysosomal vesicles from the MTOC region that is morphologically similar to that observed after inactivation of the dynein motor.