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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous secreted peptide and, in preclinical studies, preferentially induces apoptosis in tumor cells rather than in normal cells. The acquisition of resistance in cells exposed to TRAIL or its mimics limits their clinical efficacy. Because kinases are intimately involved in the regulation of apoptosis, we systematically characterized kinases involved in TRAIL signaling. Using RNA interference (RNAi) loss-of-function and cDNA overexpression screens, we identified 169 protein kinases that influenced the dynamics of TRAIL-induced apoptosis in the colon adenocarcinoma cell line DLD-1. We classified the kinases as sensitizers or resistors or modulators, depending on the effect that knockdown and overexpression had on TRAIL-induced apoptosis. Two of these kinases that were classified as resistors were PX domain-containing serine/threonine kinase (PXK) and AP2-associated kinase 1 (AAK1), which promote receptor endocytosis and may enable cells to resist TRAIL-induced apoptosis by enhancing endocytosis of the TRAIL receptors. We assembled protein interaction maps using mass spectrometry-based protein interaction analysis and quantitative phosphoproteomics. With these protein interaction maps, we modeled information flow through the networks and identified apoptosis-modifying kinases that are highly connected to regulated substrates downstream of TRAIL. The results of this analysis provide a resource of potential targets for the development of TRAIL combination therapies to selectively kill cancer cells.

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The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3␤ (GSK3␤) forms. Using model cell lines stably expressing these two GSK3␤ forms, we observed no interaction between WT GSK3␤ and HSP90, in stark contrast to KD GSK3␤ forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. As protein kinases regulate a multitude of cellular functions, it is imperative that their activity be highly regulated. This meticulous control is achieved through a variety of mechanisms, including phosphorylation, proteolytic processing, and direct engagement with other molecules, including chaperones. The heat shock protein 90 (HSP90) chaperone, in particular, is recruited to kinase clients to assist in their nascent folding and, depending on the kinase, to further contribute to its stability and function (1). A recent kinome-wide study of pairwise HSP90-kinase interactions observed various strengths of binding affinities between protein kinases and HSP90 (2), consistent with HSP90 assuming different roles depending on the client. HSP90 typically requires its cochaperone cell division cycle 37 (CDC37) to engage a kinase client, but the mechanism for kinase selection is not fully understood. There is no particular kinase class that forms stronger interactions with HSP90 (2), and there are even distinct binding specificities for closely related kinases. For example, epidermal growth factor receptor (EGFR) does not associate with HSP90, but close family member Erb-b2 receptor tyrosine kinase 2 (ERBB2) forms strong interactions with HSP90, and inhibition of HSP90 by geldanamycin induces rapid proteasome-mediated degradation of ERBB2 (3-5). Thus, amino acid sequences or structural features conserved in close family members are unlikely to be primary determinants for chaperone recognition, which is not surprising given that protein kinases represent a fraction of HSP90 clients (6). Instead, it appears that HSP90 recognizes kinases in a par...