RESUMEN
The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30-60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 microM), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Cadenas Ligeras de Miosina/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Animales , Arterias Carótidas , Colon , Femenino , Cobayas , Técnicas In Vitro , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Ratas , Quinasas p21 ActivadasAsunto(s)
Calcio/fisiología , Contracción Muscular , Músculo Liso/citología , Proteínas de Unión al GTP rho/fisiología , Animales , Calcio/química , Calcio/metabolismo , Carbacol/farmacología , Quelantes/farmacología , Compuestos de Diazonio , Femenino , Cobayas , Luz , Masculino , Modelos Biológicos , Fenoxiacetatos , Fosforilación , Fotólisis , Transporte de Proteínas , Conejos , Serina/química , Factores de Tiempo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The present study aimed to elucidate the subcellular distribution of the Cdc42 and Rac activated alpha and beta isoforms of p21-activated kinase (PAK) in the monkey cerebral neocortex and hippocampus. These proteins have been shown to play morphological roles through effects on the actin cytoskeleton. alphaPAK immunoreaction product was concentrated in regions of axon terminals or dendrites, some distance (0.2-1 microm) away from the synapse. The labeled portion of dendrite often appeared with "ruffled" cell membranes or resembling non-synapse forming "buds." betaPAK immunoreaction product was concentrated in cell bodies and larger diameter dendrites. Immunogold labeled sections showed that most of the label for both alphaPAK and betaPAK was present in a perisynaptic or extrasynaptic location, and relatively little staining was present on the postsynaptic density. Because alphaPAK has been shown to be associated with new membrane structures involving the Rho family GTPase Rac1, which controls dendritic morphology, these observations suggest alphaPAK positive regions of axons and dendrites may mark new areas of neurite extension.
Asunto(s)
Hipocampo/enzimología , Neocórtex/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Western Blotting , Células Cultivadas , Hipocampo/ultraestructura , Técnicas para Inmunoenzimas , Inmunohistoquímica , Isoenzimas/metabolismo , Macaca fascicularis , Microscopía Electrónica , Neocórtex/ultraestructura , Ratas , Fracciones Subcelulares/enzimología , Sinapsis/enzimología , Sinapsis/ultraestructura , Quinasas p21 ActivadasRESUMEN
PIX is a Rho-family guanine nucleotide exchange factor that binds PAK. We previously described two isoforms of PIX that differ in their N termini. Here, we report the identification of a new splice variant of betaPIX, designated beta2PIX, that is the dominant species in brain and that lacks the region of approximately 120 residues with predicted coiled-coil structure at the C terminus of beta1PIX. Instead, beta2PIX contains a serine-rich C terminus. To determine whether these splice variants differ in their cellular function, we studied the effect of expressing these proteins in HeLa cells. We found that the coiled-coil region plays a key role in the localization of beta1PIX to the cell periphery and is also responsible for PIX dimerization. Overexpression of beta1, but not beta2PIX, drives formation of membrane ruffles and microvillus-like structures (via activation of Rac1 and Cdc42, respectively), indicating that its function requires localized activation of these GTPases. Thus, beta1PIX, like other RhoGEFs, exerts specific morphological functions that are dependent on its intracellular location and are mediated by its C-terminal dimerization domain.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fraccionamiento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Chlorocebus aethiops , Dimerización , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Ratones , Microscopía Electrónica de Rastreo , Microvellosidades , Datos de Secuencia Molecular , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Factores de Intercambio de Guanina Nucleótido Rho , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Quinasas p21 ActivadasRESUMEN
Proteins labeled with 32P can be used as sensitive "prime" in blot overlays to detect binding proteins or domains. Small G-protein Ras can bind GTP with extremely high affinity (Kd approximately 10(-11)-10(-12) M) in the presence of Mg2+. We have taken advantage of this property of Ras to develop a vector that expresses proteins of interest such as glutathione S-transferase (GST)/Ras fusion proteins for noncovalent labeling with [gamma-32P]GTP. The labeling efficiency of this method is >60% and involves a single short incubation step. We have previously identified several binding proteins for the second SH3 domain of the adaptor Nck using this method. Here we illustrate the overlay method using the GST/Ras system and compare results with the SH3 domain labeled by phosphorylation with [gamma-32P]ATP. Both methods are similarly specific and sensitive; however, we show that signals are dependent primarily on GST-mediated probe dimerization. These dimeric probes allow a more stable probe-target complex similar to immunoglobulin interactions, thus significantly improving the sensitivity of the technique.
Asunto(s)
Western Blotting/métodos , Glutatión Transferasa/fisiología , Radioisótopos de Fósforo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Sondas Moleculares , Proteínas Oncogénicas/fisiología , Proteínas de Fusión Oncogénica/metabolismo , Radioisótopos de Fósforo/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/metabolismo , Ensayo de Unión Radioligante , Ratas , Dominios Homologos src/fisiologíaRESUMEN
BACKGROUND: Both EGF family ligands and ErbB family receptor kinases act upstream of RAS to induce mitogenesis of normal cells, such as NIH 3T3 fibroblasts. However, oncogenically mutated RAS, such as v-Ha-RAS is constitutively activated and therefore no longer requires these ligands or receptors for its activation. Nevertheless, it up-regulates the expression of these EGF family ligands. To understand the biologic significance of RAS-induced up-regulation of these ligands in both RAS-induced PAK activation and malignant transformation, we have conducted the following studies, based on the previous observations that (1) the N-terminal SH3 domain of PIX selectively binds a Pro-rich domain of 18 amino acids of PAKs, CDC42/Rac-dependent Ser/Thr kinase family, and (2) this specific interaction is essential for both PAK activation and membrane ruffling RESULTS: Using four distinct, cell-permeable, and highly specific inhibitors, namely WR-PAK18, which blocks the PAK-PIX interaction; AG 1478, which inhibits ErbB1 kinase activity; and AG 825 or AG 879, which inhibits ErbB2 kinase activity, we demonstrate that (1) the PAK-PIX interaction is essential for v-Ha-RAS-induced malignant transformation; (2) v-Ha-RAS requires not only ErbB1 but also ErbB2, which are activated through two independent autocrine pathways to induce both the PIX/Rac/CDC42-dependent PAK activation and malignant transformation in vitro; and (3) a combination of AG 879 and the Src family kinase-specific inhibitor PP1 suppresses almost completely the growth of RAS-induced sarcomas in nude mice. CONCLUSION: These findings not only change our conventional view on the role of these RAS-inducible ligands and ErbB family receptors (serving as RAS activators) but also suggest a new avenue for the treatment of RAS-associated cancers by a combination of inhibitors specific for ERbB, Src, or PAK family kinases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN , Inhibidores Enzimáticos/uso terapéutico , Glicoproteínas/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Plantas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Receptor ErbB-2/efectos de los fármacos , Tirfostinos/uso terapéutico , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Animales , Bovinos , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Femenino , Ratones , Ratones Desnudos , Fragmentos de Péptidos/farmacología , Quinazolinas , Receptor ErbB-2/fisiología , Factores de Intercambio de Guanina Nucleótido Rho , Sarcoma Experimental/tratamiento farmacológico , Tirfostinos/farmacología , Regulación hacia Arriba , Quinasas p21 Activadas , Familia-src QuinasasRESUMEN
Vimentin, one of type III intermediate filament (IF) proteins, is expressed not only in mesenchymal cells but also in most types of tumor cells. In the present study, we introduced several types of vimentin mutated at putative phosphorylation sites in its amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation induced the formation of an unusually long bridge-like IF structure between the unseparated daughter cells, although these mutants formed the filament network similar to wild type in interphase cells. Together with sites phosphorylated by Rho-kinase and protein kinase C (PKC), vimentin-Ser72, which can not be phosphorylated by any known vimentin kinase, was one of the mutation sites essential for this phenotype. We further demonstrated that vimentin-Ser72 was phosphorylated specifically at the cleavage furrow during cytokinesis. These observations suggest the existence of a novel protein kinase responsible for vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. We propose that Rho-kinase, PKC, and an unidentified vimentin-Ser72 kinase may play important roles in vimentin filament separation during cytokinesis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Mitosis , Proteínas Quinasas/metabolismo , Vimentina/genética , Vimentina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Transfección , Células Tumorales Cultivadas , Quinasas Asociadas a rhoRESUMEN
One target for the small GTPase Cdc42 is the nonreceptor tyrosine kinase activated Cdc42-associated kinase (ACK), which binds selectively to Cdc42.GTP. We report that ACK1 can associate directly with the heavy chain of clathrin. A central region in ACK1 containing a conserved motif behaves as a clathrin adaptor and competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain. Overexpressed ACK1 perturbs clathrin distribution, an activity dependent on the presence of C-terminal "adaptor" sequences that are also present in the related nonkinase gene 33. ACK1 interacts with the adaptor Nck via SH3 interactions but does not form a trimeric complex with p21-activated serine/threonine kinase, which also binds Nck. Stable low level expression of green fluorescent protein-ACK1 in NIH 3T3 cells has been used to localize ACK1 to clathrin-containing vesicles. The co-localization of ACK1 in vivo with clathrin and AP-2 indicates that it participates in trafficking, underlying an ability to increase receptor-mediated transferrin uptake.
Asunto(s)
Arrestina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Transporte Biológico , Células COS , Clatrina/metabolismo , Endocitosis , Proteínas de la Membrana/metabolismo , Ratones , Transducción de SeñalRESUMEN
The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.
Asunto(s)
Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Secuencia Conservada , Células HeLa , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina , Treonina , Transfección , Proteína de Unión al GTP cdc42 , Quinasas p21 ActivadasRESUMEN
Given the importance of the Rho GTPase family member Rac1 and the Rac1/Cdc42 effector PAK1 in T-cell activation, we investigated the requirements for their activation by the T-cell receptor (TCR). Rac1 and PAK1 activation required the tyrosine kinases ZAP-70 and Syk, but not the cytoplasmic adaptor Slp-76. Surprisingly, PAK1 was activated in the absence of the transmembrane adaptor LAT while Rac1 was not. However, efficient PAK1 activation required its binding sites for Rho GTPases and for PIX, a guanine nucleotide exchange factor for Rho GTPases. The overexpression of ssPIX that either cannot bind PAK1 or lacks GEF function blocked PAK1 activation. These data suggest that a PAK1-PIX complex is recruited to appropriate sites for activation and that PIX is required for Rho family GTPase activation upstream of PAK1. Furthermore, we detected a stable trimolecular complex of PAK1, PIX and the paxillin kinase linker p95PKL. Taken together, these data show that PAK1 contained in this trimolecular complex is activated by a novel LAT- and Slp-76-independent pathway following TCR stimulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Células Jurkat , Activación de Linfocitos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección , Proteína Tirosina Quinasa ZAP-70 , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck. PIX is a Rac GTP exchange factor that also binds the G-protein-coupled receptor kinase-interacting protein known as GIT1. Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility. This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PIX. In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events. Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain. We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Proteínas de Ciclo Celular/química , Movimiento Celular , Pollos , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Microscopía de Contraste de Fase , Modelos Biológicos , Proteínas Oncogénicas/metabolismo , Paxillin , Fosfoproteínas/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Transfección , Quinasas p21 Activadas , Proteínas de Unión al GTP rac/metabolismo , Dominios Homologos srcRESUMEN
p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.
Asunto(s)
Desmina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Desmina/química , Desmina/ultraestructura , Activación Enzimática , Guanosina Trifosfato/metabolismo , Filamentos Intermedios/química , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Péptidos y Proteínas de Señalización Intracelular , Cinética , Peso Molecular , Mapeo Peptídico , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The kinase PAK binds tightly to the SH3 domain of its partner PIX via a central proline-rich sequence. A different N-terminal sequence allows alphaPAK to bind an SH3 domain of the adaptor Nck. The Nck SH3[2] domain interacts equally with an 18-mer PAK-derived peptide and full-length alphaPAK. Detailed analysis of this binding by saturation substitution allows related Nck targets to be accurately identified from sequence characteristics alone. All Nck SH3[2] binding proteins, including PAK, NIK, synaptojanin, PRK2, and WIP, possess the motif PXXPXRXXS; in the case of PAK, serine phosphorylation at this site negatively regulates binding. We show that kinase autophosphorylation blocks binding by both Nck and PIX to alphaPAK, thus providing a mechanism to regulate PAK interactions with its SH3-containing partners. One cellular consequence of the regulatable binding of PAK is facilitation of its cycling between cytosolic and focal complex sites.
Asunto(s)
Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales , Sustitución de Aminoácidos , Humanos , Proteínas Oncogénicas/genética , Fosforilación , Análisis de Secuencia de ADN , Quinasas p21 ActivadasRESUMEN
Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Melanoma/metabolismo , Melanoma/patología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Proteína de Unión al GTP cdc42/metabolismo , Movimiento Celular , Proteína Sustrato Asociada a CrK , Activación Enzimática , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Melanoma/genética , Mutación/genética , Invasividad Neoplásica , Fosforilación , Fosfotirosina/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Tirosina Quinasas/genética , Receptores Mensajeros de Linfocitos/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Transducción de Señal , Transfección , Células Tumorales Cultivadas , Proteína de Unión al GTP cdc42/genéticaRESUMEN
The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al GTP/química , Proteínas Tirosina Quinasas/química , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada , Escherichia coli , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Proteína de Unión al GTP cdc42Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Datos de Secuencia Molecular , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Transformación Genética , Quinasas p21 ActivadasRESUMEN
Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.
Asunto(s)
AMP Cíclico/farmacología , GTP Fosfohidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Colforsina/antagonistas & inhibidores , Colforsina/farmacología , AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Activación Enzimática , Péptidos y Proteínas de Señalización Intracelular , Microinyecciones , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/metabolismo , Serina/genética , Serina/metabolismo , Trombina/farmacología , Quinasas Asociadas a rhoRESUMEN
The serine/threonine kinase p21-activated kinase (PAK) has been implicated as a downstream effector of the small GTPases Rac and Cdc42. While these GTPases evidently induce a variety of morphological changes, the role(s) of PAK remains elusive. Here we report that overexpression of betaPAK in PC12 cells induces a Rac phenotype, including cell spreading/membrane ruffling, and increased lamellipodia formation at growth cones and shafts of nerve growth factor-induced neurites. These effects are still observed in cells expressing kinase-negative or Rac/Cdc42 binding-deficient PAK mutants, indicating that kinase- and p21-binding domains are not involved. Furthermore, lamellipodia formation in all cell lines, including those expressing Rac binding-deficient PAK, is inhibited significantly by dominant-negative RacN17. Equal inhibition is achieved by blocking PAK interaction with the guanine nucleotide exchange factor PIX using a specific N-terminal PAK fragment. We conclude that PAK, via its N-terminal non-catalytic domain, acts upstream of Rac mediating lamellipodia formation through interaction with PIX.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Membrana Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Precipitación Química , Proteínas de Unión al GTP/genética , Vectores Genéticos , Nucleótidos de Guanina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Microinyecciones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Mutagénesis Sitio-Dirigida , Neuritas/fisiología , Células PC12 , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Transducción de Señal/genética , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/fisiología , Especificidad por Sustrato , Quinasas p21 Activadas , Proteínas de Unión al GTP racRESUMEN
The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.