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Abstract
The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)), AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N3[alpha-32P]ATP. The enzyme has a protein kinase activity that is Mg2+-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The Km for ATP is 81 microM. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13-acetate/L-alpha-phosphatidylserine/Ca2+ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.
Highlights
ATP Stimulation of Guanylate Cyclase Activity—To investigate the function of the photoreceptor guanylate cyclase kinase homology domain we initially tested whether ATP has any effect on guanylate cyclase activity in photoreceptor membranes
The sequence of this region [13, 14] is aligned with Ser/Thr kinases and tyrosine kinases [32,33,34] showing that 24 of the 33 highly conserved amino acids important for proper structure and function are present in the photoreceptor guanylate cyclase kinase domain
Six different high resolution crystal structures of diverse members of the protein kinase family, including the catalytic subunit of cAPK3 and that of the tyrosine kinase domain of the human insulin receptor, provide structural frameworks that predict ATP-binding sites and kinase activity (34 –39). Using this information as guidance, we undertook studies to examine whether these predicted properties are present in the photoreceptor membrane guanylate cyclase
Summary
Materials—8-N3[␣-32P]ATP was purchased from ICN. [␥-32P]ATP was purchased from Amersham Corp. Dithiothreitol, which destroys the photoactivity of 8-N3ATP, was removed from the purified guanylate cyclase using a G-50 Sephadex column equilibrated in 20 mM sodium phosphate, pH 7.0, 50 mM NaCl, 0.1% DDM, 0.025% phosphatidylcholine, and 1 g/ml each aprotinin, leupeptin, and pepstatin. Guanylate cyclase (1 g) was added to a reaction mix containing 20 M 8-N3[␣32P]ATP (2 Ci/mmol), 20 mM sodium phosphate, pH 7.0, 4 mM Na2GTP, 2.0 mM Na2ADP, 2.0 mM NaAMP, 80 mM NaCl, and 12 mM MgCl2. Kinase Reactions—Kinase assays were performed by adding enzyme to a reaction mix containing 30 M [␥-32P]ATP (ϳ5000 cpm/pmol), 20 mM MOPS, pH 7.1, 100 mM NaCl, 8 mM MgCl2, 1 mM dithiothreitol, 10 g/ml phenylmethylsulfonyl fluoride, and 1 g/ml each aprotinin, pepstatin, and leupeptin. Reaction conditions for the guanylate cyclase assay are described in the legend of Fig. 1, except for “Mn2ϩ” where 2 mM MnCl2 ϩ 1 mM MgCl2 is substituted for 6 mM MgCl2
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