Cytosolic domain of phospholamban remains associated with the Ca-ATPase following phosphorylation by cAMP-dependent protein kinase.
Cytosolic domain of phospholamban remains associated with the Ca-ATPase following phosphorylation by cAMP-dependent protein kinase.
复制标题
受 cAMP 依赖性蛋白激酶磷酸化后，受磷蛋白的胞质结构域仍与 Ca-ATP 酶相关。
DOI：
10.1111/j.1749-6632.1998.tb08281.x
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发表时间：
1998
影响因子：
5.2
通讯作者：
Squier,TC
中科院分区：
文献类型：
--
作者：
Negash,S;Sun,H;Yao,Q;Goh,SY;Bigelow,DJ;Squier,TC
SEWITE NEGASH, HONGYE SUN, QING YAO, SWEE YONG GOH, DIANA J. BIGELOW, AND THOMAS C. SQUIER* Department of Biochemistry, Cell, and Molecular Biology, University of Kansas, Lawrence, Kansas 66045-2106, USA he calcium transport activity of the Ca-ATPase in cardiac sarcoplasmic reticulum (SR) membranes is regulated by phospholamban (PLB), which represents a major target of the f-adrenergic cascade in the heart. Upon phosphorylation of PLB by cAMP-dependent protein kinase (PKA), the calcium-dependent ATPase activity is enhanced at submicromolar calcium concentrations. The inhibition of ATPase activity by PLB involves direct contact interactions between PLB and the Ca-ATPase, since the cytosolic domain of PLB has previously been shown to directly interact with a sequence near the nucleotide binding cleft of the Ca-ATPase."* Furthermore, it has been suggested that the phosphorylation of PLB may result in the dissociation of its cytosolic domain from the Ca-ATPase." There are, however, no direct measurements in biologi-cal membranes containing the Ca-ATPase of the structural alterations in the cytosolic domain of PLB upon phosphorylation by PKA. To investigate possible alterations in the physical relationship between PLB and the Ca-ATPase that correlate with the phosphorylation of PLB in biological membranes, we have coreconstituted purified preparations of the Ca-ATPase and PLB in liposomes composed of SR lipids. The calcium-dependence of the ATPase activity of the Ca-ATPase reconstituted in the presence of PLB is analogous to that observed in native cardiac SR membranes (FIG. 1). Likewise, subsequent to the phosphorylation of PLB by PKA one observes a similar activation of the Ca-ATPase in both native cardiac and reconstituted preparations, indicating that the inhibitory interaction between PLB and the Ca-ATPase in these reconstituted preparations is physiologically relevant. To di-rectly monitor the structural coupling between PLB and the Ca-ATPase, we have reconstituted a fluorescently labeled PLB containing dansyl-chloride at Lys, in either SR lipids only or in the presence of the Ca-ATPase. Dansyl-chloride was chosen, since its large Stokes shift precludes homotransfer between chromophores located on adjacent PLB monomers, and allows an unambiguous measurement of the rotational dynamics of PLB in these functionally reconstituted membranes. This fluorescent derivative of PLB inhibits the Ca-ATPase in an analogous manner to that observed in cardiac SR membranes (FIG. 1), indicating that dansyl-chloride does not interfere with either spe-
