Xu L X, Tanaka Y, Bonderenko V A, Matsuura I, Matsumoto H, Yamazaki A, Hayashi F
Department of Ophthalmology, Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Biochemistry. 1998 Apr 28;37(17):6205-13. doi: 10.1021/bi973087i.
Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
环鸟苷酸磷酸二酯酶是光转导中的关键酶，由Pαβ和两个Pγ亚基组成。Pγ与Pαβ或与转导素的α亚基（Tα）相互作用对于视网膜光感受器中cGMP磷酸二酯酶的调节至关重要。在此，我们研究了cAMP依赖性蛋白激酶对Pγ的磷酸化作用及其在体外对Pγ与Pαβ或Tα相互作用的功能影响。Pγ可被磷酸化，但与Tα复合的Pγ（GTP和GDP形式）则不能。对磷酸化Pγ中32P放射性的测量、通过激光质谱对磷酸化Pγ的分析、磷酸化氨基酸的鉴定以及Pγ突变体形式的磷酸化表明，Pγ中只有苏氨酸35被磷酸化。Pγ突变体的磷酸化还表明，调节Pαβ功能所需的Pγ的C端和N端不参与Pγ的磷酸化，但被内源性ADP核糖基转移酶进行ADP核糖基化的精氨酸33是磷酸化所必需的。磷酸化的Pγ对胰蛋白酶激活的cGMP磷酸二酯酶的抑制活性高于未磷酸化的Pγ，表明Pγ磷酸化影响了Pγ与Pαβ的相互作用。未磷酸化的Pγ抑制Tα的GTP酶活性以及水解抗性GTP类似物与Tα的结合，而Pγ磷酸化则降低了这些抑制活性。这些观察结果表明，含有苏氨酸35的Pγ结构域参与了Pγ与Tα的相互作用，并且Pγ磷酸化调节了Pγ与Tα的相互作用。我们的观察结果表明，cAMP依赖性蛋白激酶对Pγ的磷酸化可能在脊椎动物视杆光感受器的光转导调节中发挥作用。
