The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate.
To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and C delta 1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type-STaR (wt-STaR) transfectant with 1 microM PMA caused additional STa-mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A- and C delta 1029-transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt-STaR and mutants were incubated with 1 microM PMA. Subsequent 32P-radiolabeled proteins were immunoprecipitated using anti-STaR antibody, and analyzed by autoradiography after separation on SDS-PAGE. The immunoprecipitated wt-STaR but not mS1029A and C delta 1029 had a significant radioactivity. These results suggest that the effect of PMA on wt-STaR transfectants may be caused by phosphorylation of Ser1029. The C delta 1012 mutant, with further truncation (Gln1012-Phe1050) of the carboxy terminus, did not show STa-mediated GC activation. Based on these data, these 17 amino acids (Gln1012-Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.
Wada
A
A
Institute of Tropical Medicine, Nagasaki University, Japan.
Hasegawa
M
M
Matsumoto
K
K
Niidome
T
T
Kawano
Y
Y
Hidaka
Y
Y
Padilla
P I
PI
Kurazono
H
H
Shimonishi
Y
Y
Hirayama
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012694:Serine; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D009838:Oligodeoxyribonucleotides; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D012694:Serine; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D013755:Tetradecanoylphorbol Acetate