posted on 2021-03-23, 17:08authored bySvenja Wiechmann, Benjamin Ruprecht, Theresa Siekmann, Runsheng Zheng, Martin Frejno, Elena Kunold, Thomas Bajaj, Daniel P. Zolg, Stephan A. Sieber, Nils C. Gassen, Bernhard Kuster
Due
to its important roles in oncogenic signaling, AKT has been
subjected to extensive drug discovery efforts leading to small molecule
inhibitors investigated in advanced clinical trials. To better understand
how these drugs exert their therapeutic effects at the molecular level,
we combined chemoproteomic target affinity profiling using kinobeads
and phosphoproteomics to analyze the five clinical AKT inhibitors
AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib,
and MK-2206 in BT-474 breast cancer cells. Kinobead profiling identified
between four and 29 nM targets for these compounds and showed that
AKT1 and AKT2 were the only common targets. Similarly, measuring the
response of the phosphoproteome to the same inhibitors identified
∼1700 regulated phosphorylation sites, 276 of which were perturbed
by all five compounds. This analysis expanded the known AKT signaling
network by 119 phosphoproteins that may represent direct or indirect
targets of AKT. Within this new network, 41 regulated phosphorylation
sites harbor the AKT substrate motif, and recombinant kinase assays
validated 16 as novel AKT substrates. These included CEP170 and FAM83H,
suggesting a regulatory function of AKT in mitosis and cytoskeleton
organization. In addition, a specific phosphorylation pattern on the
ULK1-FIP200-ATG13-VAPB complex was found to determine the active state
of ULK1, leading to elevated autophagy in response to AKT inhibition.