Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.

The more than 500 protein kinases comprising the human kinome catalyze hundreds of thousands of phosphorylation events to regulate a diversity of cellular functions; however, the extended substrate specificity is still unknown for many of these kinases. We report here a method for quantitatively describing kinase substrate specificity using an unbiased peptide library-based approach with direct measurement of phosphorylation by tandem liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing (multiplex substrate profiling by mass spectrometry, MSP-MS). This method can be deployed with as low as 10 nM enzyme to determine activity against S/T/Y-containing peptides; additionally, label-free quantitation is used to ascertain catalytic efficiency values for individual peptide substrates in the multiplex assay. Using this approach we developed quantitative motifs for a selection of kinases from each branch of the kinome, with and without known substrates, highlighting the applicability of the method. The sensitivity of this approach is evidenced by its ability to detect phosphorylation events from nanogram quantities of immunoprecipitated material, which allows for wider applicability of this method. To increase the information content of the quantitative kinase motifs, a sublibrary approach was used to expand the testable sequence space within a peptide library of approximately 100 members for CDK1, CDK7, and CDK9. Kinetic analysis of the HIV-1 Tat (transactivator of transcription)-positive transcription elongation factor b (P-TEFb) interaction allowed for localization of the P-TEFb phosphorylation site as well as characterization of the stimulatory effect of Tat on P-TEFb catalytic efficiency.

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.