RESUMO
An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.
Assuntos
Proteínas de Transporte/metabolismo , Clonagem Molecular/métodos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteômica/métodos , Marcadores de Afinidade , Animais , Proteínas Reguladoras de Apoptose , Química Encefálica , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
Homogeneous protein-protein interaction assays without the need of a separation step are an essential tool to unravel signal transduction events in live cells. We have established an isoform specific protein kinase A (PKA) subunit interaction assay based on bioluminescence resonance energy transfer (BRET). Tagging human Ralpha(I)-, Ralpha(II)-, as well as Calpha-subunits of PKA with Renilla luciferase (Rluc) as the bioluminescent donor or with green fluorescent protein (GFP2) as the energy acceptor, respectively, allows to directly probe PKA subunit interaction in living cells as well as in total cell extracts in order to study side by side PKA type I versus type II holoenzyme dynamics. Several novel, genetically encoded cAMP sensors and-for the first time PKA type I sensors-were generated. When C- and R-subunits are assembled to the respective holoenzyme complexes inside the cell, BRET occurs with a signal up to three times above the background. An increase of endogenous cAMP levels as well as treatment with the cAMP analog 8-Br-cAMP is reflected by a dose-dependent BRET signal reduction in cells expressing wild type proteins. In contrast to type II, the dissociation of the PKA type I holoenzyme complex was never complete in cells with maximally elevated cAMP levels. Both sensors dissociated completely upon treatment with 8-Br-cAMP after cell lysis, consistent with in vitro activation assays using holoenzymes assembled from purified PKA subunits. Interestingly, incubation of cells with the PKA antagonist Rp-8-Br-cAMPS leads to a significant BRET signal increase in cells expressing PKA type I or type II isoforms, indicating a stabilization of the holoenzyme complexes in vivo. Mutant RI subunits with reduced (hRIalpha-R210K) or abolished (hRIalpha-G200E/G324E) cAMP binding capability were studied to quantify maximal signal to noise ratios for the RI-BRET sensor. Utilizing BRET we demonstrate that PKA type II holoenzyme was rendered insensitive to beta-adrenergic receptor stimulation with isoproterenol when anchoring to the plasma membrane of COS-7 cells was disrupted by either using Ht31 peptide or by depletion of membrane cholesterol.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência de Energia , Subunidades Proteicas/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Células COS , Sobrevivência Celular , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico , Holoenzimas/metabolismo , Humanos , Isoenzimas/metabolismo , Ligação Proteica , Receptores Adrenérgicos beta/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.