Kinase activity assays

In vitro activities of purified GST–NUAK1 and GST–NUAK1 [A195T] were measured using Cerenkov counting of incorporation of radioactive 32P from [γ -32P] ATP into Sakamototide substrate peptide. Reactions were carried out in a 50 μl reaction volume for 30 min at 30 μC and reactions were terminated by spotting 40 μl of the reaction mix on to P81 paper and immediately immersing in 50 mM orthophosphoric acid. Samples were washed three times in 50 mMorthophosphoric acid followed by a single acetone rinse and air drying. The kinase-mediated incorporation of [γ -32P] ATP into Sakamototide was quantified by Cerenkov counting. One unit of activity was defined as that which catalyzed the incorporation of 1 nmol of [32P]phosphate into the substrate over 1 h.

Cell lines

HEK293, MEFs and U2OS

Preparation method

Limited solubility. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

37oC

Applications

In HEK-293 cells expressing wild-type NUAK1, 3–10 μM HTH-01-015 significantly inhibits phosphorylation of MYPT1. Treatment of NUAK1+/+ MEFs with 10 μM HTH-01-015 prominently suppresses cell migration in the wound-healing assay. In U2OS cells, HTH-01-015 blocks proliferation and phosphorylation of MYPT1 to the same extent as shRNA-mediated NUAK1 knockdown.

References:

1. Sourav B, Sara J B, Hai-Tsang H, et al. Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases. Biochemical Journal, 2014, 457(1): 215-225.