RESUMEN
Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.
Asunto(s)
Melanoma/genética , Mutación , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas B-raf/genética , Proteína de Retinoblastoma/fisiología , Proteínas Supresoras de Tumor/fisiología , Quinasas raf/fisiología , Animales , Quinasa 4 Dependiente de la Ciclina/genética , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas B-raf/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.
Asunto(s)
Depresores del Apetito/farmacología , Obesidad/tratamiento farmacológico , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR gamma/agonistas , Pérdida de Peso/fisiología , Animales , Regulación del Apetito/fisiología , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/fisiopatología , PPAR alfa/genética , PPAR gamma/genética , Rosiglitazona , Tiazoles/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Animales , Ratones , Modelos Biológicos , Neoplasias/enzimología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de SeñalRESUMEN
Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha(13). Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p115 RhoGEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252--288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p115 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sphingosine 1-phosphate was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células 3T3 , Animales , Baculoviridae , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Lisofosfolípidos/farmacología , Ratones , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Eliminación de Secuencia , Esfingosina/análogos & derivados , Esfingosina/farmacología , Spodoptera , Transfección , Proteína de Unión al GTP rhoA/metabolismo , Dominios Homologos srcAsunto(s)
Endopeptidasas/análisis , Proteína Metiltransferasas/análisis , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Endopeptidasas/genética , Inhibidores Enzimáticos , Técnica del Anticuerpo Fluorescente , Humanos , Indicadores y Reactivos , Cinética , Membranas/enzimología , Datos de Secuencia Molecular , Proteína Metiltransferasas/antagonistas & inhibidores , Proteína Metiltransferasas/genética , Procesamiento Proteico-Postraduccional , Conteo por Cintilación , Homología de Secuencia de Aminoácido , Porcinos , Proteínas ras/metabolismoAsunto(s)
Perfilación de la Expresión Génica/métodos , Genes ras , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Bases de Datos Factuales , Humanos , Ratones , Fenotipo , Plásmidos , ARN/genética , Receptores de Esteroides/genética , Transcripción Genética , TransfecciónRESUMEN
Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Animales , División Celular , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Genotipo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Cinética , Mastocitos/citología , Mastocitos/metabolismo , Ratones , Ratones Mutantes , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromina 1 , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Transducción de Señal , Transfección , Proteína Activadora de GTPasa p120/metabolismoAsunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas/genética , Proteínas Recombinantes/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteína de Unión al GTP rac1/genéticaAsunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP rho , Secuencia de Aminoácidos , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Homología de Secuencia de AminoácidoRESUMEN
Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Animales , Aorta/metabolismo , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacología , Baculoviridae/metabolismo , Proteínas Sanguíneas/química , Células Cultivadas , Clonación Molecular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Guanosina Trifosfato/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Ligandos , Modelos Químicos , Mutagénesis , Nucleótidos/metabolismo , Péptidos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas Recombinantes de Fusión/metabolismo , Porcinos , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Factores de Tiempo , Proteínas ras/metabolismoRESUMEN
The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for members of the p21(ras) (Ras) family, which negatively regulates Ras output by accelerating the conversion of active Ras. GTP to inactive Ras.GDP. Analysis of tumors from patients with neurofibromatosis type 1 (NF1) has shown biochemical evidence of hyperactive Ras as well as frequent loss of the normal NF1 allele, consistent with its role as a tumor suppressor gene. Taken together, these data suggest that novel therapeutics directed against components of the Ras signaling cascade might provide effective treatments for certain pathological complications of NF1. Here we summarize data that support a role for hyperactive Ras in NF1 disease, including Ras processing, activation, and down-regulation. We review targets for rational drug design, provide preliminary results, and discuss implications for future studies. Am. J. Med. Genet. (Semin. Med. Genet.) 89:14-22, 1999.
Asunto(s)
Genes de Neurofibromatosis 1 , Neurofibromatosis 1/genética , Neurofibromatosis 1/terapia , Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Diseño de Fármacos , Proteínas Activadoras de GTPasa , Humanos , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Eliminación de Secuencia , Transducción de Señal , Proteínas Activadoras de ras GTPasa , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Compuestos de Aluminio/metabolismo , Secuencia de Aminoácidos , Animales , Fluoruros/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Factores de Intercambio de Guanina Nucleótido , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Proteínas/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de SeñalRESUMEN
Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Compuestos de Aluminio/farmacología , Animales , Células COS , Fluoruros/farmacología , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Factores de Intercambio de Guanina Nucleótido , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
A novel alteration in exon 1 of KRAS was detected by single strand conformational polymorphism analysis of DNA amplified from the bone marrow of a 4-year-old child with myeloid leukemia. Sequencing of this mutant allele revealed an insertion of three nucleotides between codons 10 and 11 resulting in an in-frame insertion of glycine. Expression of the mutant protein in NIH 3T3 cells caused cellular transformation, and expression in COS cells activated the Ras-mitogen-activated protein kinase signaling pathway. Surprisingly, Ras.GTP levels measured in COS cells established that this novel mutant accumulates to 90% in the GTP state, considerably higher than a residue 12 mutant. Biochemical analysis confirmed that the higher Ras.GTP levels correspond to a dramatic decrease in intrinsic GTP hydrolysis as well as resistance to GTPase-activating proteins. This mutation is the first dominant Ras mutation found in human cancer that does not involve residues 12, 13, or 61, and its biochemical properties should help elucidate the mechanism of oncogenic activation.
Asunto(s)
Genes ras , Leucemia Mieloide/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Células 3T3 , Enfermedad Aguda , Animales , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Transformación Celular Neoplásica , Preescolar , Activación Enzimática , Exones , Humanos , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Polimorfismo Conformacional Retorcido-Simple , Transducción de SeñalRESUMEN
The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Fosfoproteínas , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/química , Encéfalo/metabolismo , Línea Celular , Línea Celular Transformada , Cromatografía de Afinidad , Clonación Molecular , Feto , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/aislamiento & purificación , Proteínas Activadoras de GTPasa , Biblioteca de Genes , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Spodoptera , Transfección , Proteínas Activadoras de ras GTPasaRESUMEN
Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.
Asunto(s)
Células Madre Hematopoyéticas/patología , Neurofibromatosis 1/metabolismo , Proteínas/fisiología , Proteínas ras/fisiología , Animales , División Celular , Células Cultivadas , Niño , Proteínas Activadoras de GTPasa , Genes de Neurofibromatosis 1 , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Guanosina Trifosfato/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Neurofibromatosis 1/patología , Neurofibromina 1 , Proteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Activadoras de ras GTPasa , Proteínas ras/metabolismoAsunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Proteínas de Unión al GTP/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas S6 Ribosómicas , Proteínas de Unión al GTP racRESUMEN
We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains. Based on these sequences we screened a human placenta library and cloned the full-length cDNA. The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65. Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only. hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active. The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar. hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.