Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits

A. D. Wegener, Larry Jones

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Abstract

Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent M(r) = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent M(r) to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000 suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight 'monomers'. Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.

Original languageEnglish
Pages (from-to)1834-1841
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number3
StatePublished - 1984

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Phosphorylation
Sodium Dodecyl Sulfate
Proteins
Cyclic AMP-Dependent Protein Kinases
Calcium-Calmodulin-Dependent Protein Kinases
Monomers
Gels
Molecular Weight
Molecular weight
Boiling liquids
polyacrylamide gels
phospholamban
Electrophoretic mobility
Phosphoproteins
Sarcoplasmic Reticulum
Autoradiography
Protein Kinases
Canidae
Staining and Labeling

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits",
abstract = "Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent M(r) = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent M(r) to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000 suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight 'monomers'. Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.",
author = "Wegener, {A. D.} and Larry Jones",
year = "1984",
language = "English",
volume = "259",
pages = "1834--1841",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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T1 - Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits

AU - Wegener, A. D.

AU - Jones, Larry

PY - 1984

Y1 - 1984

N2 - Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent M(r) = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent M(r) to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000 suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight 'monomers'. Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.

AB - Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent M(r) = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent M(r) to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000 suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight 'monomers'. Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.

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