posted on 2018-06-19, 17:25authored byKarl W. Barber, Chad J. Miller, Jay W. Jun, Hua Jane Lou, Benjamin E. Turk, Jesse Rinehart
The
human proteome encodes >500 protein kinases and hundreds of
thousands of potential phosphorylation sites. However, the identification
of kinase–substrate pairs remains an active area of research
because the relationships between individual kinases and these phosphorylation
sites remain largely unknown. Many techniques have been established
to discover kinase substrates but are often technically challenging
to perform. Moreover, these methods frequently rely on substrate reagent
pools that do not reflect human protein sequences or are biased by
human cell line protein expression profiles. Here, we describe a new
approach called SERIOHL-KILR (serine-oriented human library–kinase
library reactions) to profile kinase substrate specificity and to
identify candidate substrates for serine kinases. Using a purified
library of >100000 serine-oriented human peptides expressed heterologously
in Escherichia coli, we perform in vitro kinase reactions to identify phosphorylated human peptide sequences
by liquid chromatography and tandem mass spectrometry. We compare
our results for protein kinase A to those of a well-established positional
scanning peptide library method, certifying that SERIOHL-KILR can
identify the same predominant motif elements as traditional techniques.
We then interrogate a small panel of cancer-associated PKCβ
mutants using our profiling protocol and observe a shift in substrate
specificity likely attributable to the loss of key polar contacts
between the kinase and its substrates. Overall, we demonstrate that
SERIOHL-KILR can rapidly identify candidate kinase substrates that
can be directly mapped to human sequences for pathway analysis. Because
this technique can be adapted for various kinase studies, we believe
that SERIOHL-KILR will have many new victims in the future.