Matsuyo Yamazaki

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase I. IDENTIFICATION OF THE KINASE AND ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VITRO

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016–15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024–15029), we showed that Pγ is phosphorylated by a previously unknown kinase (Pγ kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pγ loses its ability to interact with GTP/Tα, but gains a 10–15 times higher ability to inhibit GTP/Tα-activated PDE than that of nonphosphorylated Pγ. Thus, we propose that the Pγ phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Tα-activated PDE. Here we demonstrate that all known Pγs preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pγ kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pγ indicates that all known Pγs contain a P-X-T-P-R sequence and that this sequence is required for the Pγ phosphorylation by Pγ kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pγ kinase activity exactly coelutes with Cdk5 and p35. The Pγ kinase activity (∼85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pγ. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pγ in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pγ complexed with GTP/Tα in mature vertebrate retinal photoreceptors.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase II. ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VIVO

Retinal cGMP phosphodiesterase (PDE) is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950–32957), we have shown that all known Pγs contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pγ phosphorylation in vivo. Under dark conditions only negligible amounts of Pγ were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, ∼4% of the total Pγ was phosphorylated in less than 10 s. Pγ dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pγ phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pγ phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pγ threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pγ phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5′-O-(3-thiotriphosphate) (GTPγS) was inhibited by Pγ alone, but not by Pγ complexed with GDP/Tα or GTPγS/Tα. Under these conditions, Pγ phosphorylated by Cdk5 inhibited the light/GTPγS-activated PDE even in the presence of GTPγS/Tα. These observations suggest that phosphorylated Pγ interacts with and inhibits light/GTPγS-activated PDE, but does not interact with GTPγS/Tα in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pγ is phosphorylated by Cdk5 and the phosphorylated Pγ inhibits GTP/Tα-activated PDE, even in the presence of GTP/Tα in ROS.

Binding of cGMP to GAF Domains in Amphibian Rod Photoreceptor cGMP Phosphodiesterase (PDE) IDENTIFICATION OF GAF DOMAINS IN PDE αβ SUBUNITS AND DISTINCT DOMAINS IN THE PDE γ SUBUNIT INVOLVED IN STIMULATION OF cGMP BINDING TO GAF DOMAINS

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Pαβ) and two identical regulatory subunits (Pγ). Biochemical studies have shown that amphibian Pαβ has high affinity, cGMP-specific, non-catalytic binding sites and that Pγ stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pγ-depleted membrane-bound PDE (20–40% Pγ still present), a single type of cGMP-binding site with a relatively low affinity (K d ∼ 100 nm) was observed, and addition of Pγ increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pγ reduced its ability to stimulate cGMP binding. Among these, the site involved in Pγ phosphorylation by Cdk5 (positions 20–23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pγ inhibition of the cGMP hydrolytic activity of Pαβ. In addition, the Pγ concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Pαβ heterodimer contains two spatially and functionally distinct types of Pγ-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Pαβ-Pγ interaction in which Pγ, by binding to one of the two sites in Pαβ, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.

Residues within the Polycationic Region of cGMP Phosphodiesterase γ Subunit Crucial for the Interaction with Transducin α Subunit IDENTIFICATION BY ENDOGENOUS ADP-RIBOSYLATION AND SITE-DIRECTED MUTAGENESIS

Interaction between the γ subunit (Pγ) of cGMP phosphodiesterase and the α subunit (Tα) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pγ polycationic region to identify residues required for the interaction with Tα. Pγ, free or complexed with the αβ subunit (Pαβ) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pγ and radiolabeling of arginine-replaced mutant forms of Pγ indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pγ complexed with Tα (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Tα, was ADP-ribosylated in the presence of Tα, suggesting that a Pγ domain containing these arginines is masked by Tα. A Pγ mutant (R33,36K), as well as wild type Pγ, inhibited both GTP hydrolysis of Tα and GTP binding to Tα. Moreover, GTP-bound Tα activated Pαβ that had been inhibited by R33,36K. However, another Pγ mutant (R33,36L) could not inhibit these Tα functions. In addition, GTP-bound Tα could not activate Pαβ inhibited by R33,36L. These results indicate that a Pγ domain containing these arginines is required for its interaction with Tα, but not with Pαβ, and that positive charges in these arginines are crucial for the interaction.

A Critical Role for ATP in the Stimulation of Retinal Guanylyl Cyclase by Guanylyl Cyclase-activating Proteins*

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 ± 0.20 mm), GCAP2 enhanced the retGC activity 10–13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3–4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 ± 2.7 nm). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nm). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nm). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.