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Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK-related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis. FRNK has been proposed to compete for FAK anchoring sites in focal adhesions, but we did not detect displacement of FAK by WT-FRNK or superinhibitory S217A-FRNK. Instead, we found FRNK interacted directly with FAK, and this interaction is markedly strengthened for the superinhibitory S217A-FRNK. The results suggest that FRNK limits growth and survival signaling pathways by binding directly to FAK in an inhibitory complex, and this inhibition is relieved by phosphorylation of FRNK at S217.

To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca 2+ transient compared to the GFP control. The Ca 2+ transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca 2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with β-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.

We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH at three different sites on the cytoplasmic domain. The 6 kDa monomer bands of FlAsH-tetracysPLB showed high anisotropy (r = 0.29), reflecting null homotransfer and low mobility (S = 0.85) on the nanosecond time scale of the FlAsH fluorescence lifetime. 30 kDa bands (pentameric PLB) within the same lanes exhibited low anisotropy, suggesting intrapentameric fluorescence energy homotransfer between PLB subunits. FlAsH labels positioned at residue -6, 5, or 23 showed a graduated pattern of fluorescence depolarization corresponding to resonance energy transfer radii of 46 +/-2, 38 +/- 4, and <25 A, respectively. Pentamer anisotropy increased with heating or fluorescence photobleaching toward a maximum value similar to that determined for monomeric PLB. Fluorescence resonance energy heterotransfer was also observed in vitro and in vivo within PLB pentamers colabeled with FlAsH and the biarsenical fluorophore ReAsH. In vitro heterotransfer efficiencies were graduated by labeling position, in harmony with homotransfer results. The calculated transfer radii compare favorably to distances predicted by a computer molecular model of the phospholamban pentamer constructed from NMR solution structures. The data support a helical pinwheel model for the PLB pentamer, in which the cytoplasmic domains bend sharply outward from the central bundle of helices.