RESUMO
The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Timo/citologia , Timo/enzimologia , Animais , Cruzamento , Cálcio/fisiologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/deficiência , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cruzamentos Genéticos , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Antígeno H-Y/genética , Hemoglobinas/genética , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Timo/metabolismoRESUMO
The calcium/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is expressed in male germ cells and spermatids and has been implicated in controlling the differentiation of germ cells into mature spermatozoa. The function of CaMKIV/Gr in spermatogenesis was investigated using CaMKIV/Gr-deficient mice generated by targeted gene disruption. CaMKIV/Gr-deficient males exhibited normal spermatogenesis, and their fertility was similar to that of wild-type littermates. Notwithstanding the function of CaMKIV/Gr as an activator of cAMP response element (CRE)-dependent transcription, mRNA levels of several testis-specific CRE modulator (CREM)-regulated genes were unaltered. These results indicate that CaMKIV/Gr is not essential for spermatogenesis or for CRE-regulated gene transcription in the testis.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Isoenzimas/fisiologia , Proteínas Repressoras , Espermatogênese , Espermatozoides/fisiologia , Transcrição Gênica , Processamento Alternativo , Animais , Northern Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/deficiência , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cruzamentos Genéticos , Modulador de Elemento de Resposta do AMP Cíclico , DNA/análise , Marcação de Genes , Marcação In Situ das Extremidades Cortadas , Isoenzimas/deficiência , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/anatomia & histologiaRESUMO
X-linked autoimmunity-allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain-containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.
Assuntos
Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Hipersensibilidade Alimentar/genética , Ligação Genética/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Cromossomo X/genética , Sequência de Aminoácidos , Doenças Autoimunes/imunologia , Sequência de Bases , Diferenciação Celular , Análise Mutacional de DNA , Diabetes Mellitus Tipo 1/imunologia , Feminino , Hipersensibilidade Alimentar/imunologia , Fatores de Transcrição Forkhead , Haplótipos , Humanos , Zíper de Leucina , Masculino , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Linhagem , Estrutura Terciária de Proteína , Sítios de Splice de RNA/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Síndrome , Células Th2/citologia , Células Th2/imunologia , Fatores de Transcrição/química , Fatores de Transcrição/imunologia , Cromossomo X/imunologiaRESUMO
The Ca(2+)/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is a key effector of neuronal Ca(2+) signaling; its function was analyzed by targeted gene disruption in mice. CaMKIV/Gr-deficient mice exhibited impaired neuronal cAMP-responsive element binding protein (CREB) phosphorylation and Ca(2+)/CREB-dependent gene expression. They were also deficient in two forms of synaptic plasticity: long-term potentiation (LTP) in hippocampal CA1 neurons and a late phase of long-term depression in cerebellar Purkinje neurons. However, despite impaired LTP and CREB activation, CaMKIV/Gr-deficient mice exhibited no obvious deficits in spatial learning and memory. These results support an important role for CaMKIV/Gr in Ca(2+)-regulated neuronal gene transcription and synaptic plasticity and suggest that the contribution of other signaling pathways may spare spatial memory of CaMKIV/Gr-deficient mice.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipocampo/fisiologia , Aprendizagem em Labirinto/fisiologia , Atividade Motora/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Encéfalo/fisiologia , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/deficiência , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Estimulação Elétrica , Potenciação de Longa Duração , Masculino , Memória , Camundongos , Camundongos Knockout , Postura , Células de Purkinje/fisiologia , Células Piramidais/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , NataçãoRESUMO
Ca(2+) induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca(2+) activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca(2+)/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca(2+) activation signals and may be broadly operative in several tissues.