Neyroz P, Desdouits F, Benfenati F, Knutson J R, Greengard P, Girault J A
Dipartimento di Biochimica G. Moruzzi, Università di Bologna, Italy.
J Biol Chem. 1993 Nov 15;268(32):24022-31.
DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.
当DARPP - 32在苏氨酸34位点被环磷酸腺苷（cAMP）依赖性蛋白激酶磷酸化时，它是蛋白磷酸酶1的有效抑制剂。DARPP - 32还会在丝氨酸45和丝氨酸102位点被酪蛋白激酶II磷酸化，从而促进cAMP依赖性蛋白激酶的磷酸化作用。我们通过稳态和时间分辨荧光研究了重组大鼠DARPP - 32的构象。稳态发射光谱以及内在（色氨酸163）和外在荧光（与半胱氨酸72相连的丙烯罗丹或荧光黄）的猝灭表明这些残基完全暴露于水环境中。DARPP - 32的内在荧光分解为三个衰减成分，寿命分别为1、3.4和7纳秒，其中中间寿命成分起主要作用。变性后，与短和长衰减常数相关的振幅之比降低。通过各向异性衰减测量的DARPP - 32的旋转行为表明，色氨酸163位于高度灵活的肽链中，而半胱氨酸72则嵌入在更刚性的环境中。cAMP依赖性蛋白激酶的磷酸化不会改变任何荧光参数，而酪蛋白激酶II磷酸化仅产生轻微影响。这些发现表明DARPP - 32至少包含两个不同的结构域，并且磷酸化对其构象没有显著影响。
