RESUMO
The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependent egr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Imediatamente Precoces , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Proto-Oncogênicas , Receptores de Superfície Celular , Animais , Células CHO , Proteínas de Transporte/química , Cricetinae , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Leptina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Mutação , Fosforilação , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , RNA Mensageiro/biossíntese , Receptores para Leptina , Fator de Transcrição STAT3 , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO2 accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000-1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.
