Phosphorylation and Mutation of Phospholamban Alter Physical Interactions With the Sarcoplasmic Reticulum Calcium Pump

Abstract

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with phospholamban. In previous studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was also disordered. The functional state of phospholamban correlated with an order-to-disorder transition of phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27-to-Ala) facilitated data collection from frozen hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The two-dimensional co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.

Lethal Arg9Cys phospholamban mutation hinders Ca 2+ -ATPase regulation and phosphorylation by protein kinase A

“…We propose that these combined effects are involved in the development of DCM. An important corollary of this study is the emerging role of the PLN pentamer and the monomer-pentamer equilibrium (43). This lethal mutation revealed that oligomerization and deoligomerization of the PLN pentamer within the membrane is directly involved in the SERCA regulatory process.…”

Characterizing Phospholamban to Sarco(endo)plasmic Reticulum Ca2+-ATPase 2a (SERCA2a) Protein Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking

“…Most importantly, stimulation of Ca 2ϩ -ATPase activity and Ca 2ϩ ion transport correlated closely with inhibition of PLB cross-linking to SERCA2a, which strongly suggests that enzyme activation requires PLB dissociation. It was recently suggested that when the enzyme binds Ca 2ϩ , PLB may leave the M2/M4/M9 binding site and move to an alternate binding location in the Ca 2ϩ pump (14,19), but an accessory binding site has yet to be identified with the cross-linking approach (12, 13, 20 -25). Regardless of whether or not PLB binds at a secondary, noninhibitory site, it seems clear that the cross-linking interactions detected here and previously reflect genuine protein-protein interactions that are that closely associated with functionality of the Ca 2ϩ pump.…”

“…Techniques assessing protein-protein interactions have included co-immunoprecipitation (11), chemical crosslinking (12,13,17), FRET (14,16,18), and two-dimensional * This work was supported, in whole or in part, by National Institutes of Health Grant HL49428. 1 To whom correspondence should be addressed: Krannert Institute of Cardiology, 1800 N. Capitol Ave., Indianapolis, IN 46202. co-crystallization (2,19), which have frequently given conflicting results. For example, some studies (including our own) suggest that the Ca 2ϩ pump with bound PLB is catalytically inactive, requiring PLB dissociation prior to enzyme activation (11,12,13,15,17), whereas others maintain that PLB is a subunit of the Ca 2ϩ pump that remains attached throughout the catalytic cycle (14,16,18).…”

Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function

“…The key calcium regulatory protein in heart is sarcoplasmic/endoplasmic reticulum Ca 2 þ ATPase isoform 2a (SERCA-2a). Phospholamban (PLN) is the principal physiological inhibitor of SERCA-2a, and phosphorylation of PLN (pPLN) at residues Serine 16 or Threonine 17 enhances SERCA-2a function by decreasing the efficacy of PLN-mediated inhibition 4 . The major phosphatase-dephosphorylating PLN is protein phosphatase 1 (PP1) and the activity of this enzyme is increased in failing human hearts 5,6 .…”