A functional kinase homology domain is essential for the activity of photoreceptor guanylate cyclase 1.
Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg(2+) binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.
Bereta
Grzegorz
G
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Wang
Benlian
B
Kiser
Philip D
PD
Baehr
Wolfgang
W
Jang
Geeng-Fu
GF
Palczewski
Krzysztof
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D006152:Cyclic GMP / Q000096:biosynthesis; D055786:Gene Knockout Techniques; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008027:Light; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D011494:Protein Kinases / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism
D011956:Receptors, Cell Surface; C110715:guanylate cyclase 1; D012694:Serine; D010770:Phosphotransferases; D011494:Protein Kinases; D054648:Protein Phosphatase 2; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D008274:Magnesium