Phosphoproteomic Analysis of the Mouse Brain Cytosol Reveals a Predominance of Protein Phosphorylation in Regions of Intrinsic Sequence Disorder

Abstract

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 nonredundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phos-

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

“…S5). Intrinsically disordered regions have been hypothesized to be subject to posttranslational modifications by kinases and phosphatases, which may regulate protein function (25,61). In addition, serine 2 of CGH-1 is broadly conserved in orthologous proteins and conservation of phosphorylation at this site is supported by large-scale phosphoproteomic evidence in amphibians and mammals (62,63).…”

Control of serotonin transporter phosphorylation by conformational state

“…Although phosphorylation sites typically fall within unstructured regions and exposed loops (34), the requirement for a conformational change to facilitate accessibility to a phosphorylation site has been observed occasionally in other systems. For example, Ser51, the regulatory phosphorylation site in eukaryotic initiation factor 2α (eIF2α), is sequestered within an α-helix, but interaction with its kinase PKR promotes melting of the helix to allow access to the phosphoacceptor residue (35,36).…”

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

“…Bioinformatics analysis revealed that more than 90% of the 14-3-3 binding partners contain intrinsically disordered segments, and the majority of the high affinity 14-3-3-binding motifs are located within these disordered regions (59,60). The presence of the 14-3-3-binding motifs within flexible regions likely uncouples the binding strength from the specificity and renders these phosphorylation-dependent and highly specific interactions reversible.…”