Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase.
Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.
Tan
C M
CM
The John P. Robarts Research Institute, London, Ontario, Canada, N6A 5K8.
Kelvin
D J
DJ
Litchfield
D W
DW
Ferguson
S S
SS
Feldman
R D
RD
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology; D051381:Rats; D012694:Serine / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics; D014638:Vanadates / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019606:Phosphoamino Acids; D014638:Vanadates; D014443:Tyrosine; D012694:Serine; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D019908:Proto-Oncogene Proteins c-raf; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; D019311:Staurosporine