RESUMO
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.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas , Proteína cdc42 de Ligação ao GTP/metabolismo , Movimento Celular , Proteína Substrato Associada a Crk , Ativação Enzimática , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Melanoma/genética , Mutação/genética , Invasividade Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas Tirosina Quinases/genética , Receptores de Retorno de Linfócitos/metabolismo , Proteína p130 Retinoblastoma-Like , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Substituição de Aminoácidos , Humanos , Proteínas Oncogênicas/genética , Fosforilação , Análise de Sequência de DNA , Quinases Ativadas por p21RESUMO
Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas Heterotriméricas de Ligação ao GTP , Feromônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Epistasia Genética , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Fator de Acasalamento , Proteínas de Membrana , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Ativação Transcricional/efeitos dos fármacos , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Adesão Celular , Chlorocebus aethiops , Citoesqueleto/ultraestrutura , Primers do DNA , DNA Complementar , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/química , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Transfecção , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTPRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células COS , Proteínas de Ciclo Celular/química , Movimento Celular , Galinhas , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Glutationa Transferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Microscopia de Contraste de Fase , Modelos Biológicos , Proteínas Oncogênicas/metabolismo , Paxilina , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Transfecção , Quinases Ativadas por p21 , Proteínas rac de Ligação ao GTP/metabolismo , Domínios de Homologia de srcRESUMO
The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha resulted in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCK alpha mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization.
Assuntos
Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citoesqueleto/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Citoesqueleto/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Miotonina Proteína Quinase , Transdução de Sinais , Proteína cdc42 de Ligação ao GTPRESUMO
The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.
Assuntos
Actinas/metabolismo , Adesão Celular/fisiologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Citoesqueleto/química , Ativação Enzimática , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato) , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas Quinases/análise , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão , Transfecção , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTPRESUMO
AlphaPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129-1143, 1997) resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in alphaPAK). alphaPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding alphaPAK83-149 potently inhibited GTPgammaS-bound Cdc42-mediated kinase activation of both alphaPAK and betaPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of alphaPAK83-149 completely blocked the phenotypic effects of hyperactive alphaPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Clonagem Molecular , Citoesqueleto/enzimologia , Citoesqueleto/fisiologia , Ativação Enzimática , Escherichia coli , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão , Proteína cdc42 de Ligação ao GTPRESUMO
Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.
Assuntos
Actinas/biossíntese , Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Actinas/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Primers do DNA , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Epiderme/enzimologia , Hibridização In Situ , Integrinas/metabolismo , Dados de Sequência Molecular , Fosfotirosina/análise , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão , Homologia de Sequência de Aminoácidos , Quinases Ativadas por p21 , Proteínas rac de Ligação ao GTPRESUMO
n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Quinases , Proteínas/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Quimerina 1 , Cromossomos Humanos Par 2 , Clonagem Molecular , DNA/genética , Éxons , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase , Humanos , Masculino , Dados de Sequência Molecular , Células PC12 , Fosfatidilserinas/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcr , Mapeamento por Restrição , Alinhamento de Sequência , Espermatócitos/metabolismo , Testículo/metabolismo , Proteínas rac de Ligação ao GTP , Proteínas Ativadoras de ras GTPaseRESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Mitose , Proteínas Quinases/metabolismo , Vimentina/genética , Vimentina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Transfecção , Células Tumorais Cultivadas , Quinases Associadas a rhoRESUMO
The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.
Assuntos
Guanosina Trifosfato/farmacologia , Microtúbulos/ultraestrutura , Animais , Bovinos , Guanosina Difosfato/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.
Assuntos
Glândulas Paratireoides/análise , Hormônio Paratireóideo/análise , Animais , Anticorpos , Bovinos , Histocitoquímica , Técnicas Imunológicas , Técnicas de Imunoadsorção , Hormônio Paratireóideo/imunologia , Fragmentos de Peptídeos/imunologiaAssuntos
Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Transformação Genética , Quinases Ativadas por p21Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas/análise , Sequência de Aminoácidos , Animais , Autorradiografia/métodos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Colódio , Sequência Conservada , Eletroforese/instrumentação , Eletroforese/métodos , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/química , Proteínas Ativadoras de GTPase , Humanos , Masculino , Mamíferos , Dados de Sequência Molecular , Radioisótopos de Fósforo , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Sensibilidade e Especificidade , Sefarose , Homologia de Sequência de Aminoácidos , Testículo/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP , Proteínas rho de Ligação ao GTPAssuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Animais , Autorradiografia/métodos , Encéfalo/enzimologia , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Guanosina Difosfato/metabolismo , Humanos , Indicadores e Reagentes , Cinética , Peso Molecular , Radioisótopos de Fósforo , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Baço/enzimologia , Especificidade por Substrato , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP , Proteínas rac de Ligação ao GTPAssuntos
GTP Fosfo-Hidrolases/isolamento & purificação , Proteínas de Ligação ao GTP/isolamento & purificação , Sequência de Aminoácidos , Animais , Escherichia coli , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Biologia Molecular/métodos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/análise , Homologia de Sequência de Aminoácidos , Proteínas rho de Ligação ao GTP , Quinases Associadas a rhoAssuntos
Cálcio/fisiologia , Contração Muscular , Músculo Liso/citologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Cálcio/química , Cálcio/metabolismo , Carbacol/farmacologia , Quelantes/farmacologia , Compostos de Diazônio , Feminino , Cobaias , Luz , Masculino , Modelos Biológicos , Fenoxiacetatos , Fosforilação , Fotólise , Transporte Proteico , Coelhos , Serina/química , Fatores de Tempo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In dogs and aged 6 days or 5 months, the parathyroid (PT) glands exhibited differences of both the distribution and ultrastructure of dark and light cells. Morphometric estimates of cytoplasm, nucleus, mitochondria, rough endoplasmic reticulum (rER), Golgi apparatus, secretory granules, lysosomes and plasma membranes of both dark and light cells were obtained by point counting volumetry from PT glands of dogs of the two age groups and calculated per one hypothetical average PT cell. It was evident that in the PT glands of 6-day-old puppies the volume of the average PT cell was significantly smaller than in the PT glands of the 5-month-old dogs, but the cell surface was not significantly different. The volume of mitochondria, rER and lysosomes was higher in the average PT cell of the 5-month-old dogs, whereas the surface of Golgi apparatus and rER did not differ. Though the difference of the cellular and mitochondrial volume is probably related to the age difference between the two groups, the findings on Golgi apparatus, plasma membranes and lysosomes suggest that the secretory activity or at least the potency for the secretion of parathyroid hormone was higher in PT glands of the 6-day-old dogs than in the 5-month-old dogs.
Assuntos
Animais Recém-Nascidos/anatomia & histologia , Cães/anatomia & histologia , Glândulas Paratireoides/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Cães/crescimento & desenvolvimento , Retículo Endoplasmático/ultraestrutura , Mitocôndrias/ultraestrutura , Organoides/ultraestruturaRESUMO
Carboxypeptidase E (CPE) catalyses the removal of C-terminal basic amino acids and is implicated in the processing of peptides and hormones in secretory vesicles. The enzyme has been isolated in multiple forms from both soluble and membrane-bound compartments, and has been demonstrated to be co-secreted with peptides from pancreatic and adrenal cells. To address the question regarding the origin of the multiple forms of the enzyme, we have transfected a construct containing the cDNA for human CPE under the control of the murine-sarcoma-virus enhancer and metallothionein promoter into the C6 rat glioma cell line, which itself has extremely low levels of CPE expression. Within transfectants, multiple forms of the enzyme have been detected by Western blotting, and their sizes are comparable with that in pituitary. Fractionation of the intracellular forms of CPE with Triton X-114 at various pH values indicates that the membrane-bound, but not the soluble, form is amphipathic; this difference probably arises from post-translational modification of the enzyme. Interestingly, the secreted enzyme is found to have the amphipathic characteristics of the membrane-bound form. By modification of the cDNA sequence, enzyme lacking N-terminal and C-terminal domains have been expressed: deletion of the 'pro' region of CPE, leading to loss of the signal cleavage site, gives a more hydrophobic but active enzyme which is not efficiently secreted from the cell; deletion of a C-terminal region gives a secreted form of the enzyme which no longer exhibits amphipathic behaviour. Our data indicate that a single mRNA species can give rise to the multiple forms of CPE enzyme that have been identified and that the C6 cells are able to carry out the intracellular processing events to generate these forms.