RESUMEN
Loss of fragile X mental retardation protein FMR1 is the most common genetic cause of mental deficiency in man. We find that both FMR1 and the related FXR1 serve as direct binding partners for the Cdc42 effector PAK1. This involves an 11 residue segment in the PAK1 autoinhibitory domain that is exposed upon kinase activation and binds the FXR1 KH2 domain. Active PAK1 can phosphorylate FXR1 at Ser420; antibodies to this site show increased phosphorylation when fragile X proteins are recruited to stress granules. During zebrafish muscle development, FXR1 Ser420 phosphorylation is needed for protein function. The familial FMR1(I304N) mutation is biologically inactive, and FXR1(I304N) fails to bind PAK1. A different PAK1 binding-deficient mutant, FXR1(Q348K/E352A), fails to rescue loss of Zf-FXR1 unless combined with a gain-of-function S420D phosphomimetic. This is the first documented protein partner for the KH(2) domain of FMR1 or FXR1, and it has several implications for signaling by fragile X proteins.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Glutatión Transferasa/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Quinasas p21 Activadas/genéticaRESUMEN
PAKs (p21 activated kinases) are an important class of Rho effectors. These contain a Cdc42-Rac1 interaction and binding (CRIB) domain and a flanking auto-inhibitory domain (AID) which binds the C-terminal catalytic domain. The group II kinases PAK4 and PAK5 are considered significant therapeutic targets in cancer. Among human cancer cell lines we tested, PAK5 protein levels are much lower than those of PAK4, even in NCI-H446 which has the highest PAK5 mRNA expression. Although these two kinases are evolutionarily and structurally related, it has never been established why PAK4 is inactive whereas PAK5 has high basal activity. The AID of PAK5 is functionally indistinguishable from that of PAK4, pointing to other regions being responsible for higher activity of PAK5. Gel filtration indicates PAK4 is a monomer but PAK5 is dimeric. The central region of PAK5 (residues 109-420) is shown here to promote self-association, and an elevated activity, but has no effect on activation loop Ser(602) phosphorylation. These residues allow PAK5 to form characteristic puncta in cells, and removing sequences involved in oligomerization suppresses kinase activity. Our model suggests PAK5 self-association interferes with AID binding to the catalytic domain, thus maintaining its high activity. Further, our model explains the observation that PAK5 (1-180) inhibits PAK5 in vitro.
Asunto(s)
Multimerización de Proteína/fisiología , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Línea Celular Tumoral , Activación Enzimática/genética , Activación Enzimática/fisiología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína/genética , Quinasas p21 Activadas/genéticaRESUMEN
Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2-0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192-228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2.
Asunto(s)
Actinina/metabolismo , Actinas/metabolismo , Movimiento Celular , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas 14-3-3/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Colforsina/química , Citoesqueleto/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Metástasis de la Neoplasia , Fosforilación , Unión Proteica , Proteínas de Unión al ARN , TransfecciónRESUMEN
p21-activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1-like (group I) or PAK4-like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto-inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A-loop phosphorylation.
Asunto(s)
Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Activación Enzimática , Datos de Secuencia Molecular , Mutación , Fosforilación , Serina/metabolismo , Quinasas p21 Activadas/genéticaRESUMEN
Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, ßPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.
Asunto(s)
Plaquetas/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Quinasas p21 Activadas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Adhesión Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Quinasas Lim/genética , Quinasas Lim/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Quinasas p21 Activadas/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Rnd proteins are Rho family GTP-binding proteins with cellular functions that antagonize RhoA signaling. We recently described a new Rnd3 effector Syx, also named PLEKHG5, that interacts with Rnds via a Raf1-like "Ras-binding domain." Syx is a multidomain RhoGEF that participates in early zebrafish development. Here we demonstrated that Rnd1, Rnd2, and Rnd3 stability is acutely dependent on interaction with their effectors such as Syx or p190 RhoGAP. Although Rnd3 turnover is blocked by treatment of cells with MG132, we provide evidence that such turnover is mediated indirectly by effects on the Rnd3 effectors, rather than on Rnd3 itself, which is not significantly ubiquitinated. The minimal regions of Syx and p190 RhoGAP that bind Rnd3 are not sequence-related but have similar effects. We have identified features that allow for Rnd3 turnover including a conserved Lys-45 close to the switch I region and the C-terminal membrane-binding domain of Rnd3, which cannot be substituted by the equivalent Cdc42 CAAX sequence. By contrast, an effector binding-defective mutant of Rnd3 when overexpressed undergoes turnover at normal rates. Interestingly the activity of the RhoA-regulated kinase ROCK stimulates Rnd3 turnover. This study suggests that Rnd proteins are regulated through feedback mechanisms in cells where the level of effectors and RhoA activity influence the stability of Rnd proteins. This effector feedback behavior is analogous to the ability of ACK1 and PAK1 to prolong the lifetime of the active GTP-bound state of Cdc42 and Rac1.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Células COS , Chlorocebus aethiops , Inhibidores de Cisteína Proteinasa/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacología , Mutación , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Represoras/genética , Pez Cebra , Proteína de Unión al GTP cdc42/genética , Quinasas p21 Activadas/genética , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Quinasas p21 Activadas/química , Quinasas p21 Activadas/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía/métodos , Inhibidores Enzimáticos , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.
Asunto(s)
Proteínas 14-3-3/metabolismo , Fosfoserina/metabolismo , Mapeo de Interacción de Proteínas/métodos , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos , Sitios de Unión , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie , Fosfatasas cdc25/químicaRESUMEN
Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3ß. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Paclitaxel/farmacología , Moduladores de Tubulina/farmacología , Secuencias de Aminoácidos , Anafase/efectos de los fármacos , Anafase/fisiología , Animales , Células COS , Chlorocebus aethiops , Epotilonas/farmacología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Ratas , Semaforina-3A/genética , Semaforina-3A/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.
Asunto(s)
Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Activación Enzimática , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Pez Cebra , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to betaPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.
Asunto(s)
Movimiento Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Transcripción STAT3/metabolismo , Fibras de Estrés/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Actinas , Animales , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Transcripción STAT3/genética , Activación Transcripcional , Proteína de Unión al GTP rac1/genéticaRESUMEN
ACK1 (activated Cdc42-associated kinase 1), a cytoplsmic tyrosine kinase, is implicated in metastatic behavior, cell spreading and migration, and epidermal growth factor receptor (EGFR) signaling. The function of ACK1 in the regulation of receptor tyrosine kinases requires a C-terminal region that demonstrates a significant homology to the EGFR binding domain of MIG6. In this study, we have identified additional receptor tyrosine kinases, including Axl, leukocyte tyrosine kinase, and anaplastic lymphoma kinase, that can bind to the ACK1/MIG6 homology region. Unlike the interaction between MIG6 and EGFR, our data suggest that these receptor tyrosine kinases require the adaptor protein Grb2 for efficient binding, which interacts with highly conserved proline-rich regions that are conserved between ACK1 and MIG6. We have focused on Axl and compared how ACK1/Axl differs from the ACK1/EGFR axis by investigating effects of knockdown of endogenous ACK1. Although EGFR activation promotes ACK1 turnover, Axl activation by GAS6 does not; interestingly, the reciprocal down-regulation of GAS6-stimulated Axl is blocked by removing ACK1. Thus, ACK1 functions in part to control Axl receptor levels. Silencing of ACK1 also leads to diminished ruffling and migration in DU145 and COS7 cells upon GAS6-Axl signaling. The ability of ACK1 to modulate Axl and perhaps anaplastic lymphoma kinase (altered in anaplastic large cell lymphomas) might explain why ACK1 can promote metastatic and transformed behavior in a number of cancers.
Asunto(s)
Proteína Adaptadora GRB2/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/metabolismo , Movimiento Celular , Chlorocebus aethiops , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/clasificación , Proteína Adaptadora GRB2/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Datos de Secuencia Molecular , Proteínas Oncogénicas/clasificación , Proteínas Oncogénicas/genética , Filogenia , Proteínas Tirosina Quinasas/clasificación , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/clasificación , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Tirosina Quinasa del Receptor AxlRESUMEN
Paxillin, a major focal-adhesion complex component belongs to the subfamily of LIM domain proteins and participates in cell adhesion-mediated signal transduction. It is implicated in cell-motility responses upon activation of cell-surface receptors and can recruit, among others, the GIT1 [GRK (G-protein-coupled-receptor kinase)-interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein)]-PIX [PAK (p21-activated kinase)-interacting exchange factor]-PAK1 complex. Several adhesion proteins including zyxin, Hic5 and Trip6 are also nuclear and can exert transcriptional effects. In the present study we show that endogenous paxillin shuttles between the cytoplasm and nucleus, and we have used a variety of tagged paxillin constructs to map the nuclear export signal. This region overlaps an important LD(4) motif that binds GIT1 and FAK1 (focal-adhesion kinase 1). We provide evidence that phosphorylation of Ser(272) within LD(4) blocks nuclear export, and we show that this modification also reduces GIT1, but not FAK1, binding; however, Ser(272) phosphorylation does not appear to be mediated by PAK1 as previously suggested. Expression of nuclear-localized paxillin LIM domains stimulate DNA synthesis and cell proliferation. By real-time PCR analysis we have established that overexpression of either full-length paxillin or a truncated nuclear form suppresses expression of the parental imprinted gene H19, and modulation of this locus probably affects the rate of NIH-3T3 cell proliferation.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Paxillin/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Animales , Línea Celular , Núcleo Celular/efectos de los fármacos , Proliferación Celular , Chlorocebus aethiops , Ácidos Grasos Insaturados/farmacología , Adhesiones Focales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Paxillin/genética , FosforilaciónRESUMEN
Small GTPases are molecular switches that have been adopted to control many eukaryotic cell functions. Starting with the study of the protooncogene Ras in the early 1980s, detailed pathways have been uncovered upstream and downstream of Ras-related GTP binding proteins. Nonetheless, novel members have been discovered at a pace that has outstripped cell biologists, and thus much remains to be established regarding newer family members. Undiscovered functions are still being uncovered for "established" small GTPases such as Ras, Rho, and Ran. The topics covered at this meeting indeed demonstrate that Ras proteins are at the heart of cellular dynamics.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/fisiología , Animales , Células Eucariotas/fisiología , Proteínas Activadoras de GTPasa/fisiología , Humanos , Microtúbulos/fisiología , Transducción de Señal/fisiologíaAsunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Proteínas de Microfilamentos/metabolismo , Proteínas Quinasas/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
p21-activated kinases (PAKs) associate with a guanine nucleotide exchange factor, Pak-interacting exchange factor (PIX), which in turn binds the paxillin-associated adaptor GIT1 that targets the complex to focal adhesions. Here, a detailed structure-function analysis of GIT1 reveals how this multidomain adaptor also participates in activation of PAK. Kinase activation does not occur via Cdc42 or Rac1 GTPase binding to PAK. The ability of GIT1 to stimulate alphaPAK autophosphorylation requires the participation of the GIT N-terminal Arf-GAP domain but not Arf-GAP activity and involves phosphorylation of PAK at residues common to Cdc42-mediated activation. Thus, the activation of PAK at adhesion complexes involves a complex interplay between the kinase, Rho GTPases and protein partners that provide localization cues.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosfoproteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Activación Enzimática , Adhesiones Focales/metabolismo , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Fracciones Subcelulares/metabolismo , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase-kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.
Asunto(s)
Variación Genética/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Quinasas p21 Activadas , Quinasas Asociadas a rhoRESUMEN
A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citoplasma/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Células HeLa , Humanos , Ratones , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transporte de ProteínasRESUMEN
Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds ß integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to ß integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin ß1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Moléculas de Adhesión Celular , Proteínas del Citoesqueleto , Adhesiones Focales , Proteínas Supresoras de Tumor , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Biotinilación , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/química , Adhesiones Focales/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Sorting nexin 9 (SNX9, also referred to as SH3PX1) is a binding partner for the non-receptor and Cdc42-associated kinase (ACK) in Drosophila and mammals. ACK1 is known to bind clathrin and influence EGF receptor endocytosis. SNX9 comprises an N-terminal Src homology domain 3 (SH3), a central PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. In order to investigate SNX9 further we have made use of a novel in vivo biotinylation system to label various GST-SH3 domains and perform blot overlays, thereby identifying synaptojanin-1 as a partner for SNX9. Biotinylated SH3 domains were also used for specific identification of target proline-rich sequences in synaptojanin and ACK1 on synthetic peptides arrays. Direct assessment of SH3 binding efficiencies at different positions within the extensive proline-rich regions of these proteins were thus determined. While SNX9 targets a number of sequences within the proline-rich regions of synaptojanin, a single site was identified in human ACK1. By testing the association of various truncations of ACK1 with SNX9 we confirmed the dominant SNX9 binding domain in human ACK1 (residues 920-955). In the presence of SNX9 we find that synaptojanin is able to colocalize with distinct ACK1 containing vesicles, indicating that this tyrosine kinase is linked to many components involved in vesicle dynamics including clathrin, AP2 and synaptojanin-1.