RESUMO
The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.
Assuntos
Calcineurina/biossíntese , Calcineurina/genética , Dictyostelium/genética , Processamento Pós-Transcricional do RNA , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Calcineurina/química , Calcineurina/metabolismo , Calmodulina/metabolismo , Códon de Iniciação/genética , Dictyostelium/citologia , Dictyostelium/enzimologia , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Holoenzimas/biossíntese , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.
Assuntos
Calcineurina/isolamento & purificação , Calcineurina/metabolismo , Dictyostelium/enzimologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Compostos de Anilina/metabolismo , Animais , Calcineurina/genética , Inibidores de Calcineurina , Calmodulina/farmacologia , Caseínas/metabolismo , Cátions Bivalentes , Bovinos , Cloretos/farmacologia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , DNA Complementar , Dictyostelium/genética , Ditiotreitol/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Cloreto de Magnésio/farmacologia , Compostos de Manganês/farmacologia , Ácido Okadáico/farmacologia , Compostos Organofosforados/metabolismo , Fosfopeptídeos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão , Reagentes de Sulfidrila/farmacologia , Transformação GenéticaRESUMO
cDNA clones for the catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) from Dictyostelium discoideum were isolated by functional screening of a lambda gt11 lysogen expression library with labeled Dictyostelium CaM. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shown to display similar enzymatic properties as the enzyme from other sources. On Western blots specific antibodies against the protein recognized a band of approximately 80 kDa that migrated with an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed.