The
regulation of protein function through phosphorylation is often
dominated by allosteric interactions and conformational changes. However,
alternative mechanisms involving electrostatic interactions also regulate
protein function. In particular, phosphorylation of clusters of Ser/Thr
residues can affect protein-plasma membrane/chromatin interactions
by electrostatic interactions between phosphosites and phospholipids
or histones. Currently, only a few examples of such mechanisms are
reported, primarily because of the difficulties of detecting highly
phosphorylated proteins and peptides, due in part to the low ionization
efficiency and fragmentation yield of multiphosphorylated peptides
in mass spectrometry when using positive ion mode detection. This
difficulty in detection has resulted in under-reporting of such modified
regions, which can be thought of as phosphoproteomic dark matter.
Here, we present a novel approach that enriches for multisite-phosphorylated
peptides that until now remained inaccessible by conventional phosphoproteomics.
Our technique enables the identification of multisite-phosphorylated
regions on more than 300 proteins in both yeast and human cells and
can be used to profile changes in multisite phosphorylation upon cell
stimulation. We further characterize the role of multisite phosphorylation
for Ste20 in the yeast mating pheromone response. Mutagenesis experiments
confirmed that multisite phosphorylation of Ser/Thr-rich regions plays
an important role in the regulation of Ste20 activity during mating
pheromone signaling. The ability to detect protein multisite phosphorylation
opens new avenues to explore phosphoproteomic dark matter and to study
Ser-rich proteins that interact with binding partners through charge
pairing mechanisms
