Calcineurin B in Dictyostelium discoideum

Abstract

The genome of Dictyostelium discoideum contains a single gene (cnb1) for the regulatory (B) subunit of the Ca2+/calmodulin dependent protein phosphatase, calcineurin. 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 Mr 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´ nontranslated 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 cnb1 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 cnb1 gene.A truncated CNB cDNA fragment was recombinantly expressed in E. coli and purified. The purified protein was used for the production of polyclonal antibodies and for the biochemical studies. The biochemical datas optained are very similar to datas described for CN from other organisms. It was shown, that the recombinant CNB binds to CNA from Dictyostelium and that it increases the phosphatase activity of CNA using the RII peptide as a substrate but not when using pNPP.