Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase.
The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.
Foster
J L
JL
Department of Biochemistry, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA.
Higgins
G C
GC
Jackson
F R
FR
D000367:Age Factors; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000201:enzymology*; D004789:Enzyme Activation; D005260:Female; D007150:Immunohistochemistry; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009417:Nerve Tissue / Q000201:enzymology; D010766:Phosphorylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution
D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D017869:Cyclic GMP-Dependent Protein Kinases