Figure 1.
Large scale phosphorylation analyses add an unprecedented layer of information to kinases.
A) Regulation of enzyme activity by reversible protein phosphorylation. Because the phosphorylation state of kinases often correlates with enzymatic activity, monitoring site-specific phosphorylation events are attractive biomarkers for measuring drug-target inhibition. B) Previously reported phosphorylation sites on PDK1.
Figure 2.
Bona fide substrates are not representative of PDK1 target inhibition.
A) The T-loop phosphorylation site on PDK1Ser241 is not modulated by PDK1 inhibitor treatment in PC3 cells whereas downstream substrates, such as p-AKTThr308 and p-RSKSer221, are affected. B) In PC3 cells the inhibition of p-RSKSer221 correlates with the enzymatic potency of PDK1 inhibitors (N = 147). C) LNCap cells don't exhibit a correlation between inhibition of p-AKTThr308, a bona fide PDK1 substrate (N = 279), and the D) enzymatic potency of PDK1 inhibitors (N = 82). E) Western blot of p-AKTThr308 and p-RSKSer221 in a variety of cell lines. F) PDK1 substrates are subject to feedback regulation and pathway cross-talk.
Figure 3.
Schematic of stable isotope labeling with amino acid in cell culture and immuno-affinity precipitation for MS analysis (SILAC-IAP-MS).
T293 cells grown in either light (12C-Arg, 12C-Lys) or heavy (13C-Arg, 13C-Lys) medium were lysed and combined at a 1∶1 ratio based on total protein quantity. PDK1 was immunoprecipitated and analyzed by mass spectrometry. MS raw data were quantified by the Elucidator software 3.5 and confirmed by manual validation as described in the Materials and Methods.
Figure 4.
Relative quantification of hPDK1 phosphorylation from 293T cells treated with ATP competitive PDK1 inhibitors using SILAC-IAP-MS.
A) SDS-PAGE and isolation of the PDK1 protein band for LC-MS/MS analysis. B) PDK1 tool compounds representing three structurally distinct chemical classes. PDK1 enzymatic activity is shown (EC50). C) Mapping of phosphorylation sites of immunoprecipitated PDK1 from 293T cells identifies 12 Ser/Thr phosphorylation sites with ∼95% sequence coverage. D) Relative quantification by SILAC-IAP-MS showed a greater than 4 fold reduction of two phosphorylation sites (pS410, and pT513) in samples treated with PDK1 inhibitors compared to samples treated with DMSO. In contrast, no change in phosphorylation was detected in the self-to-self control sample where 293T cells grown in heavy and light media were both treated with DMSO prior to mixing. E) MS/MS spectra for pS241 (ANpSFVGTAQYVSPELLTEK), pS410 (DTGLPQRSGpSNIEQYIH), and pT513 (NFKpTFFVHTPNR) containing peptides.
Figure 5.
The pharmacological biomarker audit trail.
Biomarker categories demonstrate the relationship between the specificity of target engagement biomarkers (i.e. proximal biomarkers) and distal disease-related outcome biomarkers. Direct target engagement markers allow lead optimization of compounds at the kinase target level.