AAK1 Identified as an Inhibitor of Neuregulin-1/ErbB4-Dependent Neurotrophic Factor Signaling Using Integrative Chemical Genomics and Proteomics

Abstract

Summary Target identification remains a challenge for the field of chemical biology. We describe here an integrative chemical genomic and proteomic approach combining the use of differentially active analogs of small-molecule probes with stable isotope labeling by amino acids in cell culture (SILAC)-mediated affinity enrichment, followed by subsequent testing of candidate targets using RNA interference (RNAi)-mediated gene silencing. We applied this approach to characterizing the natural product K252a and its ability to potentiate neuregulin-1 (Nrg1)/ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4)-dependent neurotrophic factor signaling and neuritogenesis. We show that AAK1 (adaptor-associated kinase 1) is a relevant target of K252a, and that the loss of AAK1 alters ErbB4 trafficking and expression levels providing evidence for a previously unrecognized role for AAK1 in Nrg1-mediated neurotrophic factor signaling. Similar strategies should lead to the discovery of novel targets for therapeutic development.

Chemical Genetic Identification of NDR1/2 Kinase Substrates AAK1 and Rabin8 Uncovers Their Roles in Dendrite Arborization and Spine Development

“…How AAK1 function regulates dendrite morphogenesis remains to be investigated. Intriguingly, AAK1 was recently implicated in regulating various signaling pathways including Notch (Gupta-Rossi et al, 2011), ErbB4 (Kuai et al, 2011) and Drosophila Neuroglian (Yang et al, 2011). …”

Rapid profiling of protein kinase inhibitors by quantitative proteomics

“…We described an approach using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics and affinity matrices to quantitatively measure even small (~20%) changes in protein enrichment by SM probes over control pull-downs, allowing sensitive and specific identification of direct targets and higher-order interactors of SM probes from cell lysates 13, 14 . We applied our SILAC target ID approach to identify novel targets of immunophilin ligands 13 , natural products 15 , and kinase inhibitors 13, 16–18 . Our SILAC target ID workflow applies mild non-ionic detergents, yielding bona fide targets with one milligram of input protein and allows the identification of lower affinity interactions (K d ~ 40 µM) 13 .…”

Target identification and mechanism of action in chemical biology and drug discovery

“…An integrated approach was used to characterize the ability of a well-known natural product, K252a, to potentiate Nrg1-induced neurite outgrowth 141 . Integrating quantitative proteomic results with a lentivirus-mediated loss-of-function screen to validate candidate target proteins, the authors found that knockdown of AAK-1 reproducibly potentiated Nrg-1–driven neurite outgrowth.…”