RESUMEN
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Complejo Poro Nuclear/química , Fosfoproteínas/fisiología , Proteómica/métodos , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Carioferinas/metabolismo , Ratones , Células 3T3 NIH , Fosfoproteínas/química , Fosforilación , beta Carioferinas/metabolismoRESUMEN
Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.
Asunto(s)
Cromatografía de Afinidad/métodos , Fosfoproteínas/aislamiento & purificación , Proteoma , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Metales , Ratones , Células 3T3 NIH , Fosfoproteínas/química , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
The structure of [(Boc-Cys(1)-Pro-Leu-Cys(4)-OMe)(S-tert-C(4)H(9))Hg](-) (Boc: butoxycarbonyl), 1, was studied in N,N-dimethylformamide (DMF) and compared with that of [(Boc-Cys(1)-Pro-Leu-Cys(4)-OMe)Hg], 2, in order to discuss the intrinsic structural feature of the cysteine-containing metal-binding sites of proteins: Cys(i)-X-Y-Cys(i)(+3)/M(2+). 1 was generated by the reaction of 2 with NaS-tert-C(4)H(9). The geometry of the mercury ion (Hg(2+)) in 1 was proposed to be trigonal planar by UV-vis spectroscopy and Hg L(III) edge X-ray absorption fine structure (XAFS) measurements. Extended X-ray absorption fine structure (EXAFS) calculations yielded r(Hg-S) = 2.42 Å. Analyses of the nuclear Overhauser and exchange spectroscopy (NOESY) and the rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of 1 in DMF-d(7) gave approximate distances for the 21 (1)H-(1)H pairs of the main chain loop. These results on distance information were processed by distance geometry (DG) and restrained molecular dynamics (RMD) calculations in order to optimize the molecular structure of 1. Molecular dynamics (MD) calculations were also performed. We proposed that the trigonal planar Hg(2+) in 1 regulates the hydrogen-bonding schemes of the peptide in the same manner as the tetrahedral ions involved in the Cys(i)-X-Y-Cys(i)(+3)/M(2+) core sites in natural proteins, forming two hydrogen bonds, Cys(1) S-Leu H(N) and Cys(1) S-Cys(4) H(N). This is in contrast to 2, where the linear coordinate mercury causes another type of hydrogen-bonding scheme, Cys(1) S-Leu H(N) and Pro CO-Cys(4) H(N). Details of the effect of trigonal planar Hg(2+) on the peptide conformation were analyzed with respect to the phi, varphi, and chi torsion angles of the peptide chain. The effect of the change of the angleS-Hg-S bite angle on the conformation of Cys-Pro-Leu-Cys was also discussed on the basis of MD calculations. The distribution area of Leu (phi, varphi) in the Ramachandran plot moves from near the alpha helix region to the turn structure region as the bite angle increases from 90 to 180 degrees, accompanying the change in the hydrogen-bonding scheme. The critical bite angle is around 140 degrees. The analysis revealed that angleS-Hg-S congruent with 110 degrees, which corresponds to the tetrahedral coordination geometry of the central metal ion, allows a high flexibility of the Cys-Pro-Leu-Cys skeleton.