Abstract
A full-length cDNA corresponding to the Dictyostelium myosin light chain kinase gene has been isolated and characterized. Sequence analysis of the cDNA confirms conserved protein kinase subdomains and reveals that the Dictyostelium sequence is highly homologous to those of calcium/calmodulin-dependent protein kinases, including myosin light chain kinases from higher eukaryotes. Despite the high homologies to calcium/calmodulin-dependent protein kinases, there is no recognizable calmodulin-binding domain within the Dictyostelium sequence. However, the Dictyostelium myosin light chain kinase possesses a putative auto-inhibitory domain near its carboxyl terminus. To further characterize this domain, the full-length enzyme as well as a truncated form lacking this domain were expressed in bacterial cells and purified. The full-length enzyme expressed in bacteria exhibits essentially the same biochemical characteristics as the enzyme isolated from Dictyostelium. The truncated form however exhibits a Vmax that is approximately ten times greater than that of the native enzyme. In addition, unlike the native kinase and the full-length kinase expressed in bacteria, the truncated enzyme does not undergo autophosphorylation. These results suggest that the Dictyostelium enzyme, like myosin light chain kinases from higher eukaryotes, is regulated by an autoinhibitory domain but that the specific molecular signals necessary for activation of the Dictyostelium enzyme are entirely distinct.
Highlights
A full-length cDNA corresponding to theDictyosteZium myosin light chain kinase gene hasbeen isolated and characterized
Partial EcoRI restriction yielded within the polylinker at the 3'-end of the cDNA, and directionally an insert of approximately one kilobase that was subcloned into the bacterial expression plasmid pPLEX (Tucker et al, 1986)
In MLCKs of higher eukaryotes, the CaM-binding domain, characterized by clusters of basic and hydrophobic residues, lies carboxyl-terminal to the protein kinase catalytic domain (Blumenthal etal., 1985; Lukas et al, 1986). the corresponding region in the Dictyostelium MLCK sequence has some homology to the CaM-binding domain of higher eukaryotic MLCKs, this regiondoes not appear to satisfy the criteria of forming the basic amphiphilic a-helical structure essential for CaM bind
Summary
A full-length cDNA corresponding to theDictyosteZium myosin light chain kinase gene hasbeen isolated and characterized. These resultssuggest that the Dictyostelium enzyme, like myosin light chain kinases from higher eukaryotes, is regulated by an autoinhibitory domain but that the specific molecular signals necessary for activation of the Dictyosteliurn enzyme are entirelydistinct.
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