RESUMO
Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection. Although these effectors contribute to pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behaviour. Here we exploit two effector proteins, the Shigella flexneri OspF protein and Yersinia pestis YopH protein, to rewire kinase-mediated responses systematically both in yeast and mammalian immune cells. Bacterial effector proteins can be directed to inhibit specific mitogen-activated protein kinase pathways selectively in yeast by artificially targeting them to pathway-specific complexes. Moreover, we show that unique properties of the effectors generate new pathway behaviours: OspF, which irreversibly inactivates mitogen-activated protein kinases, was used to construct a synthetic feedback circuit that shows novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to tune the T-cell response amplitude precisely, or as an inducible pause switch that can temporarily disable T-cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.
Assuntos
Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Engenharia Genética/métodos , Sistema de Sinalização das MAP Quinases , Saccharomyces cerevisiae/enzimologia , Linfócitos T/enzimologia , Fatores de Virulência/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proliferação de Células , Células Cultivadas , Retroalimentação Fisiológica , Humanos , Interleucina-2/imunologia , Células Jurkat , Ativação Linfocitária/genética , Concentração Osmolar , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Virulência/genética , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidadeRESUMO
The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Choque Térmico , Proteômica , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Catálise , Proteína p300 Associada a E1A , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Especificidade por Substrato , Transativadores/metabolismoRESUMO
Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes.