Systematic Identification of Protein Phosphorylation-Mediated Interactions

Protein phosphorylation is a key
regulatory mechanism involved
in nearly every eukaryotic cellular process. Increasingly sensitive
mass spectrometry approaches have identified hundreds of thousands
of phosphorylation sites, but the functions of a vast majority of
these sites remain unknown, with fewer than 5% of sites currently
assigned a function. To increase our understanding of functional protein
phosphorylation we developed an approach (phospho-DIFFRAC) for identifying
the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of nonspecific
protein phosphatase treatment, size-exclusion chromatography, and
mass spectrometry allowed us to identify changes in protein interactions
after the removal of phosphate modifications. With this approach we
were able to identify 316 proteins involved in phosphorylation-sensitive
interactions. We recovered known phosphorylation-dependent interactors
such as the FACT complex and spliceosome, as well as identified novel
interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome
component ZRANB2. More generally, we find phosphorylation-dependent
interactors to be strongly enriched for RNA-binding proteins, providing
new insight into the role of phosphorylation in RNA binding. By searching
directly for phosphorylated amino acid residues in mass spectrometry
data, we identified the likely regulatory phosphosites on ZRANB2 and
FACT complex subunit SSRP1. This study provides both a method and
resource for obtaining a better understanding of the role of phosphorylation
in native macromolecular assemblies. All mass spectrometry data are
available through PRIDE (accession #PXD021422).