Hoffmann R, Wilkinson I R, McCallum J F, Engels P, Houslay M D
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson and Wolfson Buildings, IBLS, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Biochem J. 1998 Jul 1;333 ( Pt 1)(Pt 1):139-49. doi: 10.1042/bj3330139.
Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.
已证实，Ser-13和Ser-54是蛋白激酶A（PKA）介导的人cAMP特异性磷酸二酯酶同工型HSPDE4D3磷酸化的唯一位点。在RRES54的共有基序中，将Ser-54替换为带负电荷的氨基酸天冬氨酸或谷氨酸，可模拟PKA磷酸化并激活HSPDE4D3的能力。PDE4选择性抑制剂咯利普兰（4-[3-(环戊氧基)-4-甲氧基苯基]-2-吡咯烷酮）可抑制PKA磷酸化的HSPDE4D3和Ser-54→Asp突变体，其IC50值比未被PKA磷酸化的酶低约8倍。对于一系列未激活的54位残基突变体，咯利普兰抑制的IC50值较低，但具有能够作为氢键供体的侧链的突变体除外，即Ser-54→Thr、Ser-54→Tyr和Ser-54→Cys突变体。Glu-53→Ala突变体表现出与PKA磷酸化的天然酶和Ser-54→Asp突变体相当的活性，但与天然酶不同的是，尽管被这种蛋白激酶磷酸化得更快，但它对PKA激活不敏感。激活的Glu-53→Ala突变体对咯利普兰抑制的敏感性与天然酶相比没有变化。双突变体Arg-51→Ala/Arg-52→Ala的酶活性和对咯利普兰的抑制作用与天然酶相比均无变化，并且不能为PKA在Ser-54处的磷酸化提供底物。双嘧达莫对天然酶、Ser-54→Asp和Ser-54→Ala突变体的抑制作用没有差异。提出了一个模型，该模型设想PKA的磷酸化会触发HSPDE4D3中至少两种不同的构象变化；其中一种导致酶激活，另一种增强对咯利普兰抑制的敏感性。PKA介导的磷酸化激活HSPDE4D3被认为涉及破坏涉及带负电荷的Glu-53的离子对相互作用。PKA介导的磷酸化后对咯利普兰抑制敏感性的增加被认为涉及破坏涉及Ser-54侧链羟基的氢键。
