RESUMO
A series of A- and D-ring substituted estrogen analogues have been examined for their effect on estrogen receptor-alpha (ERalpha) mRNA downregulation. Recently it has been proposed that ERalpha autologous downregulation occurs via transcriptional repression exerted by the binding of the ERalpha-ligand complex to the 5' region of the coding region of the ERalpha gene. Placement of the phenolic hydroxyl group on the various carbons of the aromatic A-ring of estratrien-17betaol (carbons 1-3) produced ligands which diminished the steady state level of ERalpha mRNA in relation to their affinity for receptor. 4-Hydoxyestratrien-17betaol, on the other hand, was inactive in the downregulation of ERalpha mRNA. Although this A-ring isomer brought about apparent processing of the nuclear receptor, the ERalpha reappeared in the cytosol within 24 h. Unlike the stimulation of genes regulated via estrogen response elements, maximum autologous negative regulation of the ERalpha gene required the presence of an hydroxyl group on carbon 17 of the D-ring. These results suggest that the conformational alterations elicited in the ERalpha molecule by various ligands create surfaces capable of interacting with other transcription factors in a manner which is different when the receptor functions via a response element mechanism relative to interactions during autologous negative regulation of the ERalpha gene.
Assuntos
Regulação para Baixo , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Estradiol/metabolismo , Congêneres do Estradiol/metabolismo , Moduladores de Receptor Estrogênico/metabolismo , Receptor alfa de Estrogênio , Humanos , Ligantes , Modelos Químicos , Ligação Proteica , Receptores de Estrogênio/metabolismo , Congêneres da Testosterona/metabolismo , Células Tumorais CultivadasRESUMO
We have shown previously that GH stimulates the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal-regulated kinases) 1 and 2. To examine pathways coupling GH receptor (GHR) to MAP kinase activation, we have determined the effects of GH on SHC-growth factor receptor bound 2-son of Sevenless (SHC-Grb2-SOS) association and activation of Ras, Raf, and MAP-ERK kinase (MEK). GH promoted the rapid, transient association of SHC with the Grb2-SOS complex, which correlated with the time course of Ras, Raf, and MEK activation. Despite the continuous presence of GH, these activation events were transient with Ras, Raf, and MEK returning to near basal activity by 15 or 30 min. The inactivation of Ras, Raf, and MEK directly correlated with the serine/threonine phosphorylation of SOS and dissociation of SOS from Grb2 but not Grb2 from tyrosine-phosphorylated SHC. Phosphorylation was blocked by the MEK inhibitor, PD98059. Based upon the established functions of the MAP kinase pathway, these data indicate that GH stimulation results in the assembly of a SHC-Grb2-SOS complex that serves to activate Ras and thereby engage the Raf-MEK-ERK pathway. Activation of this pathway generates a feedback kinase cascade that phosphorylates SOS resulting in the dissociation of SHC-Grb2 complexes from SOS, thereby causing a more rapid termination of the signaling pathway than would result from SHC dephosphorylation.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno , Mitógenos/farmacologia , Proteínas Quinases/metabolismo , Receptores da Somatotropina/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Flavonoides/farmacologia , Hormônio do Crescimento/farmacologia , Humanos , MAP Quinase Quinase 1 , Proteínas de Membrana/fisiologia , Camundongos , Fosforilação , Testes de Precipitina , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-raf , Proteínas Son Of Sevenless , Fatores de Tempo , Proteínas ras/fisiologiaRESUMO
GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Hormônio do Crescimento/farmacologia , Proteínas do Leite , Proteínas Proto-Oncogênicas , Transativadores/metabolismo , Tirosina/metabolismo , Animais , Anticorpos/análise , Anticorpos/imunologia , Especificidade de Anticorpos , Western Blotting , Células CHO/citologia , Células CHO/metabolismo , Células CHO/fisiologia , Células COS/citologia , Células COS/metabolismo , Células COS/fisiologia , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/imunologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Janus Quinase 2 , Camundongos , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Ratos , Receptores da Somatotropina/genética , Fator de Transcrição STAT5 , Transativadores/imunologia , TransfecçãoRESUMO
Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains. We therefore compared the effects of CCK and epidermal growth factor (EGF) on Tyr phosphorylation of the adaptor proteins Shc and its association with Grb2 and the guanine nucleotide exchange factor SOS. Three major isoforms of Shc (p46, p52, p66) were detected in isolated rat pancreatic acini with p52 Shc being the predominant form. CCK and EGF increased tyrosyl phosphorylation of Shc (251 and 337% of control, respectively). CCK-stimulated tyrosyl phosphorylation of Shc as well as Shc-Grb2 complex formation was significant at 2.5 min, maximal at 5 min, and persisted for at least 30 min. Finally, SOS was found to be associated with Grb2 as assessed by probing of anti-Grb2 immunoprecipitates with anti-SOS. Since MAPK in pancreatic acini is activated via protein kinase C (PKC), we studied the effect of phorbol esters on Shc phosphorylation and found 12-O-tetradecanoylphorbol-13-acetate to be as potent as CCK. Furthermore, GF-109203X, a PKC inhibitor, abolished the effect of 12-O-tetradecanoylphorbol-13-acetate and also the effect of CCK but not the effect of EGF on Shc tyrosyl phosphorylation. CCK-induced tyrosyl phosphorylation of Shc was found to be phosphatidylinositol 3-kinase-independent, and CCK did not cause EGF receptor activation. These results suggest that formation of an Shc-Grb2-SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation in these cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Colecistocinina/farmacologia , Pâncreas/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Células 3T3 , Animais , Fator de Crescimento Epidérmico/farmacologia , Proteína Adaptadora GRB2 , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Camundongos , Pâncreas/citologia , Pâncreas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/metabolismo , Tirosina/metabolismo , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine kinase activity, GH has recently been shown to promote the association of its receptor with JAK2 tyrosine kinase, to activate JAK2, and to promote the tyrosyl phosphorylation of both GH receptor (GHR) and JAK2. In this work, we examined whether tyrosines 333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH. Tyrosines 333 and 338 in rat full-length (GHR1-638) and truncated (GHR1-454) receptor were replaced with phenylalanines and the mutated GHRs expressed in Chinese hamster ovary cells. These substitutions caused a loss of GH-dependent tyrosyl phosphorylation of truncated receptor and a reduction of GH-dependent phosphorylation of the full-length receptor. Consistent with Tyr333 and/or Tyr338 serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GH.GHR.JAK2 complexes. The Tyr to Phe substitutions did not substantially alter GH-dependent JAK2 association with GHR or tyrosyl phosphorylation of JAK2. These results suggest that Tyr333 and/or Tyr338 in GHR are phosphorylated in response to GH and may therefore serve as binding sites for SH2 domain-containing proteins in GH signal transduction pathways.
Assuntos
Hormônio do Crescimento/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores da Somatotropina/metabolismo , Tirosina , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Ativação Enzimática , Hormônio do Crescimento/metabolismo , Humanos , Janus Quinase 2 , Mutagênese Sítio-Dirigida , Fenilalanina , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina , Mutação Puntual , Ratos , Receptores da Somatotropina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transfecção , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Two-dimensional polyacrylamide gel-electrophoresis analysis of in vitro translation products from extracted cellular mRNAs was utilized to examine the effect of A-ring isomers of estradiol (E2) on the synthesis of proteins involved in the response of MCF-7 cells to estrogens. An 8 h pulse with 10(-8) M E2 showed 11 polypeptides of interest, 9 displayed a transient increase in mRNA accumulation and 2 showed a temporary decreased level in the presence of this hormone. A distinct set of 2 mRNAs displayed increased amounts only after a 24 h E2 pulse. Position of the A-ring hydroxyl group on the estratrien-17 beta-ol moiety had a discriminatory effect on the mRNAs for the 11 polypeptides responsive to E2. The accumulation of three mRNAs (A, C, and E) were increased by the 3 A-ring isomers (1-, 2-, and 4-hydroxyestratrien-17 beta-ol) to a degree comparable to that brought about by E2. One mRNA (H) was decreased by all estrogens. The pattern of responses depicted in the remaining 7 polypeptides was different depending on the position of the A-ring hydroxyl group of the estrogen. Subtle changes in the structure of E2 appear to attenuate the ability of this natural ligand to regulate certain estrogen responsive genes and not others. This phenomenon may be related to the interaction of TAF-2 in ligand bound receptor with the various regulators in the promoter region of specific estrogen responsive genes.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Eletroforese em Gel Bidimensional , Humanos , Estereoisomerismo , Células Tumorais CultivadasRESUMO
Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells expressing wild-type GHR (GHR1-638) or GHR truncated at amino acid 454 (GHR1-454) or 380 (GHR1-380). JAK2 did not associate with GHR in cells expressing GHR truncated at amino acid 294 (GHR1-294) or when amino acids 297-311 containing a proline-rich motif were deleted (GHR delta P) or prolines 300, 301, 303, and 305 in the proline-rich motif were mutated to alanines (GHR4P-->A). Cross-linking 125I-human GH to GHR demonstrated that GHR mutants migrated with the appropriate molecular weight, with the exception of GHR4P-->A which migrated as a protein similar in size to GHR1-294. In studies performed in CHO and RIN-5AH cells, the ability of JAK2 to associate with the mutated GHR was found to correlate with GH-dependent activation of JAK2, tyrosyl phosphorylation of GHR (in the case of GHR1-638 and GHR1-454), and the ability of the GHR to copurify with tyrosine kinase activity. In CHO cells expressing mutated GHR, GH-dependent tyrosyl phosphorylation of cellular proteins (p121, p97, p42, and p39) was dependent on the ability to activate JAK2. No proteins showed increased tyrosyl phosphorylation in CHO cells expressing GHR1-294, GHR4P-->A, or GHR delta P. Deletion of the C-terminal half (amino acids 455-638) of the GHR ablated GH-dependent tyrosyl phosphorylation of p97. Taken together, these results provide strong evidence that the N-terminal quarter of the cytoplasmic domain of GHR and within this region, the proline-rich motif, is required for association of JAK2 with GHR and GH-dependent activation of JAK2, and that tyrosines in the N-terminal half of the cytoplasmic domain of the GHR are phosphorylated by JAK2. The finding that a specific interaction with the C-terminal half of GHR appears to be necessary for p97 phosphorylation indicates that while JAK2 activation may be necessary for a full biological response to GH, it appears not to be sufficient.
Assuntos
Hormônio do Crescimento/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Análise Mutacional de DNA , Ativação Enzimática , Humanos , Janus Quinase 2 , Fosforilação , Ligação Proteica , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue. E2 induced CAT expression at levels as low as 10(-13) M, with maximum induction at 10(-11) M. CAT activity decreased at higher concentrations of E2. Estratriene, which has low affinity for ER, was active only at micromolar concentrations. 3-Hydroxyestratriene displayed maximal activity at 10(-9) M, with higher levels being less active. Still higher concentrations (10(-7) M) of estratrien-17 beta-ol were required to induce maximum CAT activity. All positional and conformational alterations in the D-ring hydroxyl group of E2 yielded active ligands. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring produced dihydroxyestrogens with varied capacities to activate CAT (2-hydroxyestratrien-17 beta-ol produced maximum CAT activation at 10(-11) M; 1-hydroxyestratrien-17 beta-ol required a 10(-8) M concentration for maximum activity; 4-hydroxyestratrien-17 beta-ol gave maximum CAT activation at 10(-6) M). Only those androstanediols or 5-androstenediols with a 3 beta-hydroxyl group were capable of activating CAT expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Estradiol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Estradiol/análogos & derivados , Estradiol/metabolismo , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , TATA Box , Transfecção , Células Tumorais CultivadasRESUMO
The relationship between steroid structure, estrogen receptor (ER) binding affinity, nuclear binding of the ER complex, and induction of progesterone receptor (PgR) have been examined. The level of ER in membrane-free homogenates of MCF-7 cells was found to be 10.0 +/- 0.5 fmol/micrograms of DNA by utilizing an enzyme immunoassay (EIA). However, only 2.5 +/- 0.2 fmol of ER complex/micrograms of DNA was bound by nuclei during maximal stimulation of PgR synthesis (2.9 +/- 0.2 fmol of PgR/micrograms of DNA; measured by EIA) following a pulse with 10(-10) M E2. Except at micromolar concentrations, estratriene was an ineffective estrogen. The addition of a hydroxyl group to either position 3 or position 17 beta of estratriene yielded ligands which were capable of causing nuclear binding and processing of ER as well as PgR induction. D-ring regioisomers of estradiol (E2) had lower affinity for receptor than E2. However, receptor complexed with these estrogens was fully capable of binding to nuclear material, undergoing processing, and inducing PgR. On the other hand, A-ring regioisomers of E2 displayed significant differences in their ability to mediate nuclear binding of receptor complex and induction of PgR. Although 1-hydroxyestratrien-17 beta-ol was weakly bound by ER, this dihydroxyestrogen was capable of bringing about nuclear binding and processing of ER and the stimulation of PgR synthesis. In contrast, 2- and 4-hydroxyestratrien-17 beta-ol, which caused extensive nuclear binding of ER (5-7 fmol/micrograms of DNA), were incapable of significant PgR induction.(ABSTRACT TRUNCATED AT 250 WORDS)