Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In
order to understand cellular signaling, a clear understanding
of kinase–substrate relationships is essential. Some of these
relationships are defined by consensus recognition motifs present
in substrates making them amendable for phosphorylation by designated
kinases. Here, we explore a method that is based on two sequential
steps of strong cation exchange chromatography combined with differential
stable isotope labeling, to define kinase consensus motifs with high
accuracy. We demonstrate the value of our method by evaluating the
motifs of two very distinct kinases: cAMP regulated protein kinase
A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as
TTK. PKA is a well-studied basophilic kinase with a relatively well-defined
motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation,
has been less well characterized. Its substrate specificity is unclear
and here we show that Mps1 is an acidophilic kinase with a striking
tendency for phosphorylation of threonines. The final outcomes of
our work are high-definition kinase consensus motifs for PKA and Mps1.
Our generic method, which makes use of proteolytic cell lysates as
a source for peptide-substrate libraries, can be implemented for any
kinase present in the kinome.