Protein phosphorylation is one of the most important and prevalent PTMs and is involved in regulation of diverse cellular functions, including cell division, growth, differentiation, and intercellular communication. [1
] Protein kinases typically catalyze phosphorylation of serine, threonine, or tyrosine residues by transferring the gamma phosphate group of ATP to substrate proteins. The family of protein kinases – the kinome – is encoded by approximately 518 ORFs in humans and by 122 ORFs in the budding yeast. [3
] The proteins comprising the kinome have been considered to be highly attractive targets for drug development in a wide variety of diseases. [6
Previous studies have indicated that in addition to phosphorylation, the activities and functions of protein kinases are regulated by diverse types of PTMs, such as ubiquitination, acetylation, glycosylation, and myristoylation. [7
] Nevertheless, identities of PTMs other than phosphorylation have not been carefully examined in kinases. [12
] A comprehensive description of the full-spectrum of PTMs is required to further characterize protein kinase functions, and to elucidate the mechanisms by which kinase pathways in biological processes are regulated.
Here, we report a systemic study of the non-phosphorylation PTMs in protein kinases (). Our strategy combines three main steps to achieve high-confidence and full-spectrum PTM identification. First, GST-tagged yeast kinases were affinity-purified and resolved on SDS-PAGE. Second, the kinase proteins were in-gel digested and analyzed by nano-HPLC/MS/MS. Finally, MS/MS were aligned with corresponding protein sequences using PTMap, we recently developed an algorithm for unrestrictive identification of all possible PTMs, whether known or unknown. Using this strategy, we identified 53 non-phosphorylation PTM sites in 30 yeast kinases. In addition to acetylation and methylation, some previously unknown mass shifts were also found, suggesting possible novel PTMs. Our results show that the complexity of PTMs in kinases is more diverse than previously thought. Our study also demonstrates that this strategy allows for the discovery of novel, unknown PTMs with low stoichiometry.
Schemes for PTM analysis of yeast kinases.