Site-specific phosphorylation of PSD-95 dynamically regulates the postsynaptic density as observed by phase separation
Maria Vistrup-Parry,
Xudong Chen,
Thea L. Johansen,
Sofie Bach,
Sara C. Buch-Larsen,
Christian R.O. Bartling,
Chenxue Ma,
Louise S. Clemmensen,
Michael L. Nielsen,
Mingjie Zhang,
Kristian Strømgaard
Affiliations
Maria Vistrup-Parry
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark
Xudong Chen
Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Thea L. Johansen
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark
Sofie Bach
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark
Sara C. Buch-Larsen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Christian R.O. Bartling
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark
Chenxue Ma
Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Louise S. Clemmensen
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark
Michael L. Nielsen
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
Mingjie Zhang
Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; Shenzhen Bay Laboratory, Gaoke Innovation Center, Guangqiao Road, Guangming District, Shenzhen, China
Kristian Strømgaard
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark; Corresponding author
Summary: Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem, and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD.
