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• W2026963354 abstract "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. 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. cGMP phosphodiesterase the α and β subunits of PDE the γ subunit of PDE rod outer segments the α and βγ subunits of transducin the GTP-bound form of Tα guanosine 5′-O-(3-thiotriphosphate) phosphatidylinositol protein kinase C protein kinase A cyclin-dependent protein kinase a neuronal activator of Cdk5 a proteolytic digested form of p35 polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride dithiothreitol mitogen-activated protein kinase 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid The hydrolysis of cGMP by PDE1 in vertebrate ROS is directly involved in visual signal transduction (1Hurley J.B. Annu. Rev. Physiol. 1987; 49: 793-812Crossref PubMed Google Scholar, 2Yau K.-W. Baylor D.A. Annu. Rev. Neurosci. 1989; 12: 289-327Crossref PubMed Scopus (436) Google Scholar). The inactive PDE is composed of Pαβ, catalytic subunits, and two Pγs, regulatory subunits (3Miki N. Baraban J.M. Keirns J.J. Boyce J.J. Bitensky M.W. J. Biol. Chem. 1975; 250: 6320-6327Abstract Full Text PDF PubMed Google Scholar, 4Baehr W. Devlin M.J. Applebury M.L. J. Biol. Chem. 1979; 254: 11669-11677Abstract Full Text PDF PubMed Google Scholar, 5Deterre P. Bigay J. Forquet F. Robert M. Chabre M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2424-2428Crossref PubMed Scopus (167) Google Scholar, 6Fung B.K.-K. Young J.H. Yamane H.K. Griswold-Prenner I. Biochemistry. 1990; 29: 2657-2664Crossref PubMed Scopus (81) Google Scholar). In amphibian ROS, PDE is regulated similarly to that in mammalian ROS (7Fung B.K.-K. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1982; 78: 152-156Crossref Scopus (513) Google Scholar, 8Hurley J.B. Stryer L. J. Biol. Chem. 1982; 257: 11094-11099Abstract Full Text PDF PubMed Google Scholar): PDE catalytic activity is controlled by Pγ and GTP/Tα. In frog ROS membranes, bleached rhodopsin stimulates GTP/GDP exchange on Tα (9Yamazaki A. Tatsumi M. Torney D.C. Bitensky M.W. J. Biol. Chem. 1987; 262: 9316-9323Abstract Full Text PDF PubMed Google Scholar), and the GTP/Tα formed is released from membrane-bound Tβγ (9Yamazaki A. Tatsumi M. Torney D.C. Bitensky M.W. J. Biol. Chem. 1987; 262: 9316-9323Abstract Full Text PDF PubMed Google Scholar, 10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). The free GTP/Tα interacts with Pαβγγ, and Pγ complexed with GTP/Tα is released from Pαβ/membranes (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar, 11Yamazaki A. Stein P.J. Chernoff N. Bitensky M.W. J. Biol. Chem. 1983; 258: 8188-8194Abstract Full Text PDF PubMed Google Scholar, 12Arshavsky V.Y. Dumke C.L. Bownds M.D. J. Biol. Chem. 1992; 267: 24501-24507Abstract Full Text PDF PubMed Google Scholar). PDE is thereby activated. The release of the Pγ complex is detected even in an isotonic buffer containing Mg2+, and Pγ complexed with GTPγS/Tα can be isolated using sequential column chromatography (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). During PDE activation, Pγ-less Pαβ binds tightly to membranes. 2V. A. Bondarenko, M. Yamazaki, and A. Yamazaki, unpublished observations. In the recovery processes of frog ROS, after GTP hydrolysis by Tα, Pγ remains in the complex with GDP/Tα (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). When the GDP/Tα/Pγ complex interacts with membrane-bound Tβγ, Pγ is released from the complex and reassociates with Pαβ, resulting in the turnoff of PDE (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). The Pγ complex with GDP/Tα is very tight, and the GDP/Tα/Pγ complex can be isolated by sequential column chromatography (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). It has been suggested that Pγ phosphorylation is involved in the PDE regulatory mechanism. Pγ is phosphorylated by PI-stimulated kinase (13Hayashi F. Lin G.Y. Matsumoto H. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4333-4337Crossref PubMed Scopus (31) Google Scholar), PKC (14Udovichenko I.P. Cunnick J. Gonzalez K. Takemoto D.J. J. Biol. Chem. 1994; 269: 9850-9856Abstract Full Text PDF PubMed Google Scholar), PKA (15Xu L.X. Tanaka Y. Bondarenko V.A. Matsuura I. Matsumoto H. Yamazaki A. Hayashi F. Biochemistry. 1998; 37: 6205-6213Crossref PubMed Scopus (28) Google Scholar), and Pγ kinase (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar, 17Tsuboi S. Matsumoto H. Yamazaki A. J. Biol. Chem. 1994; 269: 15016-15023Abstract Full Text PDF PubMed Google Scholar). In the PI-dependent Pγ phosphorylation (13Hayashi F. Lin G.Y. Matsumoto H. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4333-4337Crossref PubMed Scopus (31) Google Scholar), threonine 35 or serine 40 in Pγ may be phosphorylated. PKC (14Udovichenko I.P. Cunnick J. Gonzalez K. Takemoto D.J. J. Biol. Chem. 1994; 269: 9850-9856Abstract Full Text PDF PubMed Google Scholar) and PKA (15Xu L.X. Tanaka Y. Bondarenko V.A. Matsuura I. Matsumoto H. Yamazaki A. Hayashi F. Biochemistry. 1998; 37: 6205-6213Crossref PubMed Scopus (28) Google Scholar) phosphorylates threonine 35 in Pγ. The phosphorylated Pγ has a higher inhibitory activity against GTP/Tα-activated PDE than that of nonphosphorylated Pγ. The important point in the Pγ phosphorylations by these protein kinases is that the Pγ phosphorylations appear not to occur when Pγ binds to GTPγS/Tα. We have shown that Pγ phosphorylations by PI-stimulated kinase (13Hayashi F. Lin G.Y. Matsumoto H. Yamazaki A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4333-4337Crossref PubMed Scopus (31) Google Scholar) and PKA (15Xu L.X. Tanaka Y. Bondarenko V.A. Matsuura I. Matsumoto H. Yamazaki A. Hayashi F. Biochemistry. 1998; 37: 6205-6213Crossref PubMed Scopus (28) Google Scholar) were inhibited by GTPγS/Tα. In the case of Pγ phosphorylation by PKA (15Xu L.X. Tanaka Y. Bondarenko V.A. Matsuura I. Matsumoto H. Yamazaki A. Hayashi F. Biochemistry. 1998; 37: 6205-6213Crossref PubMed Scopus (28) Google Scholar), the inhibition is due to the unavailability of the phosphorylation site in Pγ, because a Pγ region, including threonine 35, is involved in its interaction with GTP/Tα, and the region is masked when Pγ is complexed with GTP/Tα. The same kind of inhibition was also observed in the ADP-ribosylation of Pγ, because the Pγ ADP-ribosylation site (arginines 33 or 36) is masked when Pγ is complexed with GTP/Tα (18Bondarenko V.A. Desai M. Dua S. Yamazaki M. Amin R.H. Yousif K.K. Kinumi T. Ohashi M. Komori N. Matsumoto H. Jackson K.W. Hayashi F. Usukura J. Lipkin V.M. Yamazaki A. J. Biol. Chem. 1997; 272: 15856-15864Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Bondarenko V.A. Yamazaki M. Hayashi F. Yamazaki A. Biochemistry. 1999; 38: 7755-7763Crossref PubMed Scopus (13) Google Scholar). It is very likely that the phosphorylation of threonine 35 in Pγ occurs when Pγ is complexed with Pαβ. We have shown that arginine 33 or 36 in Pγ is ADP-ribosylated when Pγ is complexed with Pαβ (18Bondarenko V.A. Desai M. Dua S. Yamazaki M. Amin R.H. Yousif K.K. Kinumi T. Ohashi M. Komori N. Matsumoto H. Jackson K.W. Hayashi F. Usukura J. Lipkin V.M. Yamazaki A. J. Biol. Chem. 1997; 272: 15856-15864Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In contrast to these Pγ phosphorylations, Pγ phosphorylation by Pγ kinase appears to be light-dependent and thus can be brought into agreement with in the current model of phototransduction (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar, 17Tsuboi S. Matsumoto H. Yamazaki A. J. Biol. Chem. 1994; 269: 15016-15023Abstract Full Text PDF PubMed Google Scholar). In the phosphorylation, Pγ complexed with GTP/Tα is the best substrate for Pγ kinase, and the Pγ phosphorylation is dependent upon GTP in ROS membranes. These results indicate that the Pγ phosphorylation occurs after PDE activation. Threonine 22 in Pγ is phosphorylated. The phosphorylated Pγ loses its affinity to GTP/Tα, but gains a 10∼15 times higher ability to inhibit PDE activity than that of nonphosphorylated Pγ. Thus, the phosphorylated Pγ more effectively inhibits GTP/Tα-activated PDE than nonphosphorylated Pγ, and the inhibition occurs even in the presence of GTP/Tα. These observations imply that 1) the Pγ phosphorylation is probably involved in the recovery phase of phototransduction to the dark state, 2) after activation of PDE, GTP/Tα may interact with another effector and the interaction may be associated with mechanisms for the recovery of phototransduction, and 3) the lifetime of GTP/Tα-activated PDE can be regulated by the Pγ phosphorylation when the Pγ phosphorylation functions. In this series of experiments, we showed that Cdk5 phosphorylates Pγ complexed with GTP/Tα in vitro (in the accompanying paper (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar)) and in vivo (in this paper). In the accompanying paper (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we have shown that Pγ preserves an amino acid sequence required for the phosphorylation by Cdk5 and that the Pγ kinase is Cdk5 complexed with p35, a Cdk5 activator. We have also demonstrated that recombinant Cdk5/p35 phosphorylates Pγ in a GTPγS-dependent manner in ROS membranes, suggesting that Cdk5 is involved in the phosphorylation of Pγ complexed with GTP/Tα. In the present study, we link these observations with light-dependent Pγ phosphorylation in vivo(21Hayashi F. FEBS Lett. 1994; 338: 203-206Crossref PubMed Scopus (7) Google Scholar). Using frog photoreceptor outer segments isolated by a new method, we show that Cdk5 is involved in the light-dependent Pγ phosphorylation in vivo, that the Pγ phosphorylation is significant and reversible, and that the Pγ phosphorylation and dephosphorylation are rapid enough to be involved in the recovery phase of phototransduction. Moreover, in an homogenate of photoreceptor outer segments, the phosphorylated Pγ inhibits light/GTPγS-activated PDE, even in the presence of GTPγS/Tα. These observations suggest that the Pγ phosphorylation verified in the in vitro system (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar, 17Tsuboi S. Matsumoto H. Yamazaki A. J. Biol. Chem. 1994; 269: 15016-15023Abstract Full Text PDF PubMed Google Scholar, 20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) functions similarly in functional, isolated photoreceptor outer segments. Chemical reagents were purchased from the following sources: [γ-33P]ATP, [γ-32P]ATP, [3H]cGMP and [33P]phosphorus from NEN Life Science Products; ATP, cGMP, GTP, and GTPγS from Roche Molecular Biochemicals; phosphocreatine, creatine phosphokinase, PMSF, leupeptin, pepstatin A, aprotinin, olomoucine, roscovitine, iso-olomoucine, and PI from Sigma; okadaic acid from LC Services; molecular sieve (4A 1/16) from Wako Pure Chemicals; Immobiline DryStrip gels, a chemiluminescence detection kit, and Pharmalyte (pH 8–10.5) from Amersham Pharmacia Biotech; nitrocellulose membranes (0.2 μ) and Bio-Lytes (pH 5–7 and pH 6–8) from Bio-Rad; andl-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (sequencing grade) from Promega. Cdk5 and p35 antibodies used (Santa Cruz Biotechnology) were the same as those used in a previous study described in the accompanying paper (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Characterization of these antibodies was also described in the accompanying paper (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The MAP kinase antibody used (New England Biolabs) was prepared against peptides corresponding 350–360 amino acids of human p44 MAP kinase. A Pγ antibody was prepared using a peptide corresponding to bovine Pγ Arg24–Gly46. This antibody recognizes bovine and frog Pγ. All experiments described were carried out using frogs (Rana catesbiana). Recombinant bovine Cdk5/p35 was prepared by a Mono S column (22Qi Z. Huang Q.-Q. Lee K.-Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Cdk5 and p35 cloned from bovine retina have the same cDNA sequences as those of bovine brain proteins (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We note that recombinant p35 is expressed as a ∼25-kDa protein, as described previously (22Qi Z. Huang Q.-Q. Lee K.-Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Tang D.M. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 24Ishiguro K. Kobayashi O. Omori A. Takamatsu M. Yonekura S. Auzai K. Imahori K. Uchida T. FEBS Lett. 1994; 342: 203-208Crossref PubMed Scopus (149) Google Scholar). The ∼25-kDa protein (p25) is a truncated form of p35, but p25 has been shown to activate Cdk5. Pγ kinase was isolated from a soluble fraction of frog ROS using a Mono Q column (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar). Frog Pγ (10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar) and its phosphorylated form (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar) were isolated as described. Recombinant bovine Pγ was prepared as described previously (18Bondarenko V.A. Desai M. Dua S. Yamazaki M. Amin R.H. Yousif K.K. Kinumi T. Ohashi M. Komori N. Matsumoto H. Jackson K.W. Hayashi F. Usukura J. Lipkin V.M. Yamazaki A. J. Biol. Chem. 1997; 272: 15856-15864Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Frog GDP/Tα was purified as described previously (9Yamazaki A. Tatsumi M. Torney D.C. Bitensky M.W. J. Biol. Chem. 1987; 262: 9316-9323Abstract Full Text PDF PubMed Google Scholar, 10Yamazaki A. Hayashi F. Tatsumi M. Bitensky M.W. George J.S. J. Biol. Chem. 1990; 265: 11539-11548Abstract Full Text PDF PubMed Google Scholar). GTPγS/Tα was prepared from GDP/Tα by using GTPγS, urea-treated ROS membranes, and Tβγ, as described previously (9Yamazaki A. Tatsumi M. Torney D.C. Bitensky M.W. J. Biol. Chem. 1987; 262: 9316-9323Abstract Full Text PDF PubMed Google Scholar). To prepare Pγ complexed with GTPγS/Tα or GDP/Tα, these Tαs were incubated with equimolar concentration of Pγ at 4 °C overnight, and these complexes were isolated by gel-filtration columns. Frogs were fully dark-adapted (>12 h) at room temperature. Before bleaching samples, all manipulations were done under infrared light. Photoreceptor outer segments were isolated as described previously (19Bondarenko V.A. Yamazaki M. Hayashi F. Yamazaki A. Biochemistry. 1999; 38: 7755-7763Crossref PubMed Scopus (13) Google Scholar). Briefly, the frontal hemisphere of an eyeball was removed by a razor blade, and the resulting eye-cup was vertically cut in half. The half-eye-cup was put on two layers of curved filter paper (Toyo Filter Paper 5B, 3 × 6 cm). After its vitreous body was soaked by the filter papers, the retina layer adhered to the filter paper. The retina could be peeled off from the eye-cup when the curved filter paper was flattened. The pigment epithelium layer stayed on the eye-cup. The retina was covered with 100 μl of Ringer's solution (105 mm NaCl, 2.5 mm KCl, 1.2 mmMgCl2, 10 mm HEPES (pH 7.5), 2 mmtaurine, and 5 mm glucose) containing [33P]phosphorus (∼1 mCi/ml) and incubated for 30 min under O2/CO2 (95:5) atmosphere. [33P]Phosphorus was used to avoid illumination of rhodopsin by the Cerenkov effect of [32P]phosphorus. The pH value of the incubation medium was exactly adjusted to 7.50 by the addition of 0.1 n NaOH. After rinsing with Ringer's solution, the retina was placed on a flat end of a plastic plunger, and the excess Ringer's solution was removed by dry filter paper. The retina on the plastic plunger was exposed to white light (2% rhodopsin bleached/min) for the indicated periods and quickly frozen by attaching its photoreceptor outer segment layer to the surface of a copper block pretreated with liquid N2. The retina was moved into cold acetone (−20 °C, 100 ml) containing 5 g of molecular sieve (4A 1/16) and incubated (−20 °C, 3 days) with acetone. The acetone was replaced each day. Acetone was removed by decantation, and dehydrated retinas were dried under vacuum. A piece of an adhesive tape (Scotch 3M) was attached to the outer segment surface of the retina layer. After the backing filter paper was removed, another piece of tape was attached to the neural retina layer. By pulling these two tapes apart, the outer segment layer and the neural retina layer, including the photoreceptor inner segments, were separated. The outer segment layer attached to the tape is observed by light microscopy (× 500) (Fig. 1 A). The purity of the outer segments will be described later. The outer segment layer was solubilized with 100 μl of buffer A (1% Triton X-100, 1% deoxycholic acid, 150 mmNaCl, 50 mm Tris/HCl (pH 7.5), 1 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 50 nm okadaic acid) containing 1% SDS, heated at 95 °C for 5 min, and diluted with 900 μl of buffer A. Insoluble materials were spun down by ultracentrifugation. The protein concentration was determined by the bicinchoninic acid protein assay method (25Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar) after proteins were precipitated with deoxycholic acid/trichloroacetic acid. Pγ and phosphorylated Pγ in each sample (1.0 mg of protein) were immunoprecipitated using a Pγ-specific antibody and further purified by SDS-PAGE. Radioactive bands corresponding to phosphorylated Pγ were detected by using an image analyzer and were cut out. Phosphoamino acid analysis of the Pγ was carried out as described previously (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar). 33P-Labeled spots were detected on an image analyzer. Radioactive spots were recovered, and their radioactivities were measured. The purity of photoreceptor outer segments isolated was investigated using antibodies against MAP kinase, which are believed not to be present in outer segments (20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Photoreceptor outer segments and the neural retinal layer were solubilized in 200 μl of SDS-sample buffer containing 70 mm DTT and heated at 95 °C for 5 min. Proteins (20 μg) in these samples were isolated by SDS-PAGE, and MAP kinase was detected by Western blot. The protein concentration was measured as described previously (25Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Fresh frog retina was immediately fixed with 4% paraformaldehyde in 0.13m phosphate buffer (pH 7.4) for 2 h at 4 °C. After washing with the same buffer (×3), the retina was equilibrated with 30% sucrose solution. Cryosections (14 μm thick) were prepared with a Cryostat (Leica CM3050 Bensheim, Germany), and mounted on glass slides. These specimens were blocked with the phosphate-buffered saline buffer containing 1% (w/v) bovine serum albumin and 0.5% Triton X-100 for 30 min at room temperature. Sections were incubated with a Cdk5 antibody at the IgG concentration of 1 ng/ml for 2 h. For controls, the same concentration of the antibody was mixed with the peptide antigen (20 ng/ml) prior to application. Specimens were incubated with a secondary antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG (15 mg/ml), for 1 h. These specimens were washed with the phosphate buffer (×3). Finally, antibody binding sites were visualized with 5-bromo-4-chloro-3′-indolyl phosphatep-toluidine salt and nitro blue tetrazolium chloride. Before illumination of samples, all manipulations were done under infrared light. Retinas were isolated from frog eye-cups as described above. These retinas were incubated in Ringer's solution (20 min). After exposure to white light (2% rhodopsin bleached/min) for indicated times, these retinas were quickly frozen as described above. As a control, all procedures were carried out without light. The photoreceptor outer segment layer was isolated from dried retina as described above and then solubilized with 50 μl of 1% SDS containing 100 mm DTT. Solubilized outer segment sample was diluted by 10-fold with buffer B (2% Triton X-100, 1% Pharmalyte (pH 8–10.5), 0.5% Bio-Lyte (pH 5–7), 0.5% Bio-Lyte (pH 6–8), 1 mmPMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 100 nm okadaic acid, and 9.2 m urea in final concentrations). Pγ and phosphorylated Pγ in the solubilized outer segments (50 μg protein) were isolated by two-dimensional gel electrophoresis using Immobiline DryStrip gels, consisting of isoelectric focusing (pH 6–11) in the first dimension and SDS-gradient gel (10–20%) electrophoresis in the second dimension. Pγ and phosphorylated Pγ were blotted on nitrocellulose membranes, and membranes were blocked by 5% milk in Tris-buffered saline containing 0.1% Tween 20. A Pγ-specific antibody diluted in the same blocking buffer was used to identify Pγ, and the bound antibody was detected by a chemiluminescence detection kit. After development of x-ray films, Pγ spots were scanned by Paragon 1200A3 ProScanner and relative density (mm2 × OD) was calculated by Molecular Analyst software (Bio-Rad). The location of phosphorylated Pγ in gels was confirmed using 33P-phosphorylated Pγ. In the in vivo system, Cdk inhibitor, olomoucine (100 μm), or roscovitine (50 μm) were applied to retinas incubated in Ringer's solution containing [33P]phosphorus. Other experimental conditions were the same as those described above. An inactive analogue of olomoucine, iso-olomoucine (100 μm), was used as a control. Isolation of Pγ and phosphoamino acid analysis of phosphorylated Pγ were carried out as described above. Effects of these inhibitors on Pγ kinase and recombinant Cdk5/p35 were also measured using Pγ phosphorylation in vitro (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar, 20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Phosphopeptide maps of Pγ phosphorylatedin vitro and in vivo were compared. As Pγ phosphorylated in vitro, frog Pγ (10 μg) was phosphorylated by using ∼10 μCi of [γ-33P]ATP and Pγ kinase, as described previously (16Tsuboi S. Matsumoto H. Jackson K.W. Tsujimoto K. Williams T. Yamazaki A. J. Biol. Chem. 1994; 269: 15024-15029Abstract Full Text PDF PubMed Google Scholar, 20Matsuura I. Bondarenko V.A. Maeda T. Kachi S. Yamazaki M. Usukura J. Hayashi F. Yamazaki A. J. Biol. Chem. 2000; 275: 32950-32957Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As Pγ phosphorylated in vivo, after incubation of retinas in Ringer's solution containing [33P]phosphorus, Pγ was phosphorylated by 10-min light exposure (20% rhodopsin was bleached), as described above. These phosphorylated Pγs were isolated by immunoprecipitation using a Pγ-specific antibody and SDS-PAGE. Pγ radioactive spots were identified by autoradiography and cut out. Extracted Pγs were digested with trypsin, and the resulting peptides were analyzed using two-dimensional peptide map analysis as described previously (26Geer P.D. Luo K. Scfton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. Oxford University Press, New York1995: 31-59Goog" @default.
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• W2026963354 title "Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase" @default.
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