Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase

We have used fluorescence and spin-label EPR spectroscopy to investigate ho w the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kina se (PKA) modifies structural interactions between PLB and the Ca2+- and Mg2 +-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl ('ITC -TEMPO'), we have co-reconstituted PLB with affinity-purified Ca-ATPase iso lated from skeletal sarcoplasmic reticulum (SR) with full retention of cata lytic function. The Ca2+-dependence of the ATPase activity of this reconsti tuted preparation is virtually identical with that observed using native ca rdiac SR before and after PLB phosphorylation, indicating that co-reconstit uted sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1 (SERCA1) and PLB prov ide an equivalent experimental model for SERCA2a-PLB interactions. Phosphor ylation of PLB in the absence of the Ca-ATPase results in a greater amplitu de of rotational mobility, suggesting that the structural linkage between t he transmembrane region and the N-terminus is destabilized. However, wherea s co-reconstitution with the Ca-ATPase restricts the amplitude of rotationa l motion of PLB, subsequent phosphorylation of PLB does not significantly a lter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PL B are retained following the phosphorylation of PLB by PKA. On the other ha nd, the fluorescence intensity decay of bound dansyl is sensitive to the ph osphorylation state of PLB, indicating that there are changes in the tertia ry structure of PLB coincident with enzyme activation. These results sugges t that PLB phosphorylation alters its structural interactions with the Ca-A TPase by inducing structural rearrangements between PLB and the Ca-ATPase w ithin a defined complex that modulates Ca2+-transport function.