RESUMO
BACKGROUND/AIMS: The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. METHODS: In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. RESULTS: We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. CONCLUSION: Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.
Assuntos
Cálcio/metabolismo , Núcleo Celular/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/enzimologia , Citoplasma/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/genética , Sistema de Sinalização das MAP Quinases , Espectrometria de Massas , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , RatosRESUMO
The subcellular localization of ERKs in cells, which is important for proper signaling, may be regulated through protein-protein interactions. We found that inactive ERK2 interacts with a large number of proteins through its cytosolic retention sequence/common docking domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERK interactions also influenced the localization of ERKs, as calcium chelators enhanced nuclear translocation, whereas elevated calcium levels prevented it. This effect of calcium was apparent upon lysophosphatidic acid stimulation, where ERKs translocation was delayed compared with that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERK translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Upon stimulation, ERKs are phosphorylated and released from cytoplasmic anchors to allow shuttling toward the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and, therefore, also their spatiotemporal regulation. Thus, calcium regulates ERK localization, which is important for the compartmentalization of ERKs with their proper substrates and thereby their signaling specificity.
