Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding
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Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding. / Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik; Madsen, Kenneth Lindegaard.
In: Biochemistry, Vol. 51, No. 2, 17.01.2012, p. 586-96.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding
AU - Ammendrup-Johnsen, Ina
AU - Thorsen, Thor Seneca
AU - Gether, Ulrik
AU - Madsen, Kenneth Lindegaard
PY - 2012/1/17
Y1 - 2012/1/17
N2 - PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.
AB - PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.
KW - Amino Acid Substitution
KW - Animals
KW - Binding Sites
KW - COS Cells
KW - Calcium-Calmodulin-Dependent Protein Kinase Type 2
KW - Carrier Proteins
KW - Cell Membrane
KW - Cercopithecus aethiops
KW - Lipid Metabolism
KW - Models, Molecular
KW - Mutagenesis, Site-Directed
KW - Mutation
KW - Nuclear Proteins
KW - PDZ Domains
KW - Phosphorylation
KW - Protein Kinase C-alpha
KW - Protein Transport
KW - Serine
U2 - 10.1021/bi2014689
DO - 10.1021/bi2014689
M3 - Journal article
C2 - 22129425
VL - 51
SP - 586
EP - 596
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 2
ER -
ID: 46375150