RESUMO
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Catálise , Linhagem Celular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Quinases Ativadas por p21/genéticaRESUMO
Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (â¼9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA3, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from â¼0.3 ns (unstructured) to â¼0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs.
Assuntos
Algoritmos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas/química , Proteínas/ultraestrutura , Conformação Proteica , Coloração e Rotulagem/métodosRESUMO
Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.
Assuntos
Escherichia coli/metabolismo , Transdução de Sinais , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Glioblastoma/patologia , Células HEK293 , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por SubstratoRESUMO
Biochemical investigation of protein phosphorylation events is limited by inefficient production of the phosphorylated and non-phosphorylated forms of full-length proteins. Here using a genomically recoded strain of E. coli with a flexible UAG codon we produce site-specific serine- or phosphoserine-containing proteins, with purities approaching 90%, from a single recombinant DNA. Specifically, we synthesize human MEK1 kinase with two serines or two phosphoserines, from one DNA template, and demonstrate programmable kinase activity. Programmable protein phosphorylation is poised to help reveal the structural and functional information encoded in the phosphoproteome.
Assuntos
Códon de Terminação/genética , Escherichia coli/genética , Genoma Bacteriano/genética , MAP Quinase Quinase 1/genética , Fosforilação/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , MAP Quinase Quinase 1/biossíntese , Organismos Geneticamente Modificados , Fosfosserina , SerinaRESUMO
Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signalling networks and kinase inhibitor drugs.
Assuntos
Sistema Livre de Células , MAP Quinase Quinase 1/biossíntese , Fosfoproteínas/biossíntese , Fosfosserina/metabolismo , Proteínas Recombinantes/biossíntese , Western Blotting , Ensaios Enzimáticos , Escherichia coli , Proteínas de Fluorescência Verde , Humanos , MAP Quinase Quinase 1/metabolismo , Espectrometria de Massas , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas , Proteínas Recombinantes/metabolismoRESUMO
Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA3 is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA3 has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h((+1))/h(0), the local tumbling volume (V(L)), and the percent change of the h(â1) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h((+1)/)h(0) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA3. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA3. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.