Phosphorylation and dephosphorylation of purified phospholamban and associated phosphatidylinositides

Abstract

Phospholamban, the putative regulator for the calcium pump, was purified to apparent homogeneity and in high yields from canine cardiac sarcoplasmic reticulum membranes. Purified phospholamban migrated with an apparent Mr of 27,000 in alkaline sodium dodecyl sulfate-polyacrylamide gels, and upon boiling in 7.5% sodium dodecyl sulfate, it dissociated into a lower molecular weight component of 5500-6000. Purified phospholamban contained 0.62 +/- 0.09 mumol of lipid Pi/mg of protein, and the major phospholipids were phosphatidylserine (34%), phosphatidylcholine (22%), sphingomyelin (17%), phosphatidylinositol (13%), and phosphatidylethanolamine (9%). Phospholamban was phosphorylated by cAMP-dependent protein kinase to a level of 207 nmol of Pi/mg, and this would indicate an incorporation of 1 mol of phosphate/mol of protein, assuming a molecular weight of 5500 for phospholamban. Phosphorylation of phospholamban could be reversed by a "phospholamban phosphatase" isolated from canine cardiac cytosol. Phospholipids associated with the purified phospholamban were also phosphorylated in the presence of the catalytic subunit of cAMP-dependent protein kinase, and the maximal phosphate incorporation was 4 nmol/mg of protein. The main phospholipids phosphorylated were phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. Phosphorylation of phospholipids was inhibited by the heat-stable inhibitor protein of the cAMP-dependent protein kinase, and it could be also reversed by the phospholamban phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban

“…Ca2"-transport ATPases and phospholamban a possible role of phosphorylated phospholipids in addition to PLB phosphorylation in the stimulation of the Ca2+-ATPase in cardiac SR [34]. In summary, our data suggest that the extended C-terminal tail of the SERCA2b Ca2l pump may be responsible for its higher Ca2+ affinity compared with the truncated SERCA2a variant.…”

Biochemical and Biophysical Comparison of Native and Chemically Synthesized Phospholamban and a Monomeric Phospholamban Analog

“…PLB was purified from cardiac SR using previously described extraction and solubilization procedures (24). The solubilized protein in Zwittergent 3-14 was applied to a mAb 1D11 immunoaffinity column with a 3.3-ml bed volume pre-equilibrated with column buffer (10 mM MOPS/KOH, pH 7.0, 0.2% Zwittergent 3-14).…”

“…Because it is critical to verify the biochemical integrity of s-PLB by comparison to native protein, sufficient quantities of highly purified n-PLB are necessary to establish its biochemical properties. The use of a 1D11 immunoaffinity column facilitated the purification of n-PLB by dramatically decreasing isolation time and improving the yield of highly purified protein as compared with procedures using sulfhydryl affinity chromatography (21,22,24). The immunoaffinity column proved to be very efficient at purifying solubilized PLB because more than 90% of the PLB bound to the column was recovered.…”

Regulation of the Skeletal Sarcoplasmic Reticulum Ca2+Pump by Phospholamban in Reconstituted Phospholipid Vesicles

“…Phospholamban was purified from cardiac SR by a modification of the method of Jakab and Kranias (7). Zwittergent 3-14 (for the cross-linking studies) or N-octylglucoside (for the reconstitution studies) was used to solubilize the final dialysate and to elute phospholamban from the column (10).…”