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

ABSTRACT: We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca(2+)- and Mg(2+)-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 isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca(2+)-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a-PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca(2+)-transport function.

SUBMITTER: Negash S

PROVIDER: S-EPMC1221350 | biostudies-other | 2000 Oct

REPOSITORIES: biostudies-other

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Project description:Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca(2+)/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca(2+)/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525-529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. (32)P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca(2+)/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca(2+)/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2-3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.

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

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