Phosphorylation of the recombinant spliced variants of the alpha-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (Gs) by the catalytic sub-unit of protein kinase A.
Both GS alpha-1 and GS alpha-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A. Phosphate incorporation into 220 pmol and 190 pmol of GS alpha-4 and GS alpha-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively. These low levels of phosphorylation are due to the thermal lability of purified recombinant GS alpha. However, the phosphorylation was inhibited by guanine nucleotides (GDP-beta-S, GppNHp and GTP) and is, therefore, a specific event. We suggest that, as for GS alpha phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of GS alpha is the most likely substrate. Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free GS alpha. GS alpha phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of GS alpha may be an important regulatory event in cells.
Pyne
N J
NJ
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, U.K.
Freissmuth
M
M
Pyne
S
S
D001345:Autoradiography; D001921:Brain / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014644:Genetic Variation; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D012322:RNA Precursors; D011994:Recombinant Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D011494:Protein Kinases; D019204:GTP-Binding Proteins