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Abstract
Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha 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 Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma 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 Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.
Highlights
Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction
It has been believed that cGMP binding to noncatalytic sites on mammalian P␣ is not regulated by P␥, because the cGMP binding to P␣ has been shown to be very tight [23]
We found that the P␥ mutant in which threonine 22 was replaced by valine (T22V) was not phosphorylated by P␥ kinase; the P␥ mutant in which threonine 35 was substituted by valine (T35V) was phosphorylated (Fig. 1)
Summary
Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. Recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates P␥ in frog outer segment membranes in a GTP-dependent manner These observations suggest that Cdk is probably involved in the recovery phase of phototransduction through phosphorylation of P␥ complexed with GTP/T␣ in mature vertebrate retinal photoreceptors. Amphibian P␥ functions as an inhibitor of cGMP hydrolysis by P␣ and as a stimulator of cGMP binding to noncatalytic sites on P␣ These functionally different P␥s appear to be released by GTP/T␣ in different steps of phototransduction [16]. A crucial point of this P␥ phosphorylation is that P␥ complexed with GTP/T␣ is the best substrate for P␥ kinase and that P␥ kinase phosphorylates P␥ in ROS membranes in a GTP-dependent manner These observations indicate that the P␥ phosphorylation occurs only after PDE activation. 5-CCTTTCCTGGGGACGACGGGTCCCC-3ЈЈ 5-CCTTTCCTGGGGTCGACGGGTCCC-3Ј Ј 5-CCTTTCCTGGGCTCGACGGGTCCC-3Ј 5-CCTTTCCTGGGCTTGACGGGTCCC-3Ј 5-GAACTGCCTCACTTGCCGCTGC-3Ј 5-GGGAGTGACGCCTCCCCCCATCAC-3Ј 5-CCCTTTCCTGCCAGTGACGGGTCC-3ЈЈ 5-CCCTTTCCTTCCAGTGACCCCTCC-3Ј 5-CCGTCACTCCCAAAAAAGGGCCCCC-3Ј 5-CCGTCACTCCCCACAAAGGGCCCCC-3ЈЈ 5-CCGTCACTCCGGAGAAAGGGCCC-3ЈЈ 5-TCACTCCCAGGATCGGGCCCCCGAA-3ЈЈ 5-TCACTCCCAGGCACGGGCCCCCGAA-3ЈЈ 5-TCACTCCCAGGAGCGGGCCCCCGAA-3ЈЈ a Underlined letters indicate mutation sites. b These mutants have been used previously [30]
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