Identification of novel phosphorylation sites on postsynaptic density proteins.
Phosphorylation of the components of the postsynaptic density (PSD), a protein complex lining the postsynaptic membrane, may regulate synaptic structure and function. We carried out mass spectrometric analyses to identify phosphorylation sites on PSD proteins. Phosphopeptides were isolated from the total tryptic digest of a PSD fraction by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The phosphorylated residues detected following in vitro phosphorylation in the presence of Ca2+/calmodulin included S-1058 on SynGAP and S-1662 and S-1668 on Shank3. Other phosphorylated residues were identified in control samples, presumably reflecting phosphorylation in the intact cell. These included the homologous residues, S-295 on PSD-95 and S-365 on PSD-93, located between the PDZ2 and PDZ3 domains of these proteins; and S-367 located on the actin-binding domain of beta-CaMKII. The sequence RXXSPV emerged as a common phosphorylation motif of three specialized PSD scaffolding proteins, PSD-95, PSD-93, and Shank3. Phosphorylated serine residues in several of the identified phosphorylation sites were followed by prolines, suggesting prominent involvement of proline directed kinases in the regulation of PSD components.
Jaffe
H
H
Protein and Peptide Sequencing Facility, NIH/NINDS, Bethesda, MD, USA.
Vinade
L
L
Dosemeci
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C501451:Mpp2 protein, rat; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; C033652:postsynaptic density proteins; D010768:Phosphoserine; D000255:Adenosine Triphosphate