Kinase Assays

Kinase assays for PKN3 were performed using the synthetic peptide GGGGPKGPGRRGRRRTSSFAEGG as sub-strate.⁵⁰ Assay conditions were 26 mM Tris pH 7.5, 0.002% CHAPS, 5.2 mM MgCl₂, 2.3 mM DTT, 25 ng PKN3, 20 μM substrate, 68 μM ATP, 1 μCi ATP [γ-³²P]. Enzyme was preincubated with compounds for 30 min before adding ATP and reacting for 30 min at room temperature. Reaction was stopped with TCA and spotted in 96 well phosphocellulose plates (Millipore). Wells were washed seven times with 150 mM phosphoric acid, once with 95% isopropanol, and dried before scintillation counting. For the time-course PKN3 kinase assay data in Figure 2C, all compound treatments were at 1 μM and preincubation times were from 0 to 120 min. Kinase assays for PRKCQ, CDK7, LIMK1, PKN1, and PKN2 were performed using SelectScreen (Invitrogen) in a 10-dose titration format.

For in vitro validation of PKN3 substrates, the following changes were made to the above methodology: candidate substrate synthetic peptides were used at 4 μM concentration, ATP was used at 1 mM concentration, and ATP [γ−³²P] was omitted. Reactions were performed for 1 h at 37 °C and stopped by adding acetic acid to 1% FC. Ten pmol of each substrate peptide was desalted and mixed with α-cyano-4-hydroxycinnamic acid matrix and analyzed on a 4800 MALDI TOF/TOF (AB SCIEX). Unphosphorylated peptide signal was compared to a control reaction lacking ATP using label free quantitation to indirectly determine the percentage of phosphopeptide conversion. Synthetic peptide sequences: LAD1 S375, RSSPRTISFRMKPKK; EXOC2 S432, ASLKRGSSFQSGRDDTWR; EGFR S1064, NGLQSCPIKEDSFLQRYSSDPT.