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1.
J Biol Chem ; 277(30): 27294-304, 2002 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-12004056

RESUMO

A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Mesoderma/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Domínio Catalítico , Ciclo Celular , Movimento Celular , Cromatografia Líquida de Alta Pressão , Cruzamentos Genéticos , Fibroblastos/metabolismo , Vetores Genéticos/metabolismo , Genótipo , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Ligação Proteica , Isoformas de Proteínas , Transdução de Sinais , Fatores de Tempo , Transfecção
2.
Mol Endocrinol ; 16(3): 630-9, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11875122

RESUMO

The intracellular second messenger cAMP affects cell physiology by directly interacting with effector molecules that include cyclic nucleotide-gated ion channels, cAMP-regulated G protein exchange factors, and cAMP-dependent protein kinases (PKA). Two catalytic subunits, Calpha and Cbeta, are expressed in the mouse and mediate the effects of PKA. We generated a null mutation in the major catalytic subunit of PKA, Calpha, and observed early postnatal lethality in the majority of Calpha knockout mice. Surprisingly, a small percentage of Calpha knockout mice, although runted, survived to adulthood. This growth retardation was not due to decreased GH production but did correlate with a reduction in IGF-I mRNA in the liver and diminished production of the major urinary proteins in kidney. The survival of Calpha knockout mice after birth is dependent on the genetic background as well as environmental factors, but sufficient adult animals were obtained to characterize the mutants. In these animals, compensatory increases in Cbeta levels occurred in brain whereas many tissues, including skeletal muscle, heart, and sperm, contained less than 10% of the normal PKA activity. Analysis of sperm in Calpha knockout males revealed that spermatogenesis progressed normally but that mature sperm had defective forward motility.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/deficiência , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Transtornos do Crescimento/enzimologia , Infertilidade Masculina/enzimologia , Espermatozoides/fisiologia , Animais , Encéfalo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fator de Crescimento Insulin-Like I/genética , Rim/metabolismo , Fígado/química , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Mutagênese , Miocárdio/enzimologia , Biossíntese de Proteínas , RNA Mensageiro/análise , Motilidade dos Espermatozoides/genética , Espermatogênese/genética , Espermatozoides/enzimologia
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