Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding - PubMed (original) (raw)

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding

David W Powell et al. Mol Cell Biol. 2003 Aug.

Abstract

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

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Figures

FIG. 1.

FIG. 1.

Recombinant MAPKAPK2 phosphorylation of neutrophil lysate. Candidate MAPKAPK2 substrates were identified by phosphorylation of neutrophil lysates with [32P]ATP in the presence and absence of recombinant active MAPKAPK2. Proteins were separated by SDS-PAGE, and phosphoproteins were detected by comparing autoradiographs to Coomassie blue-stained gels. No endogenous kinase activity resulted from incubation of neutrophil lysate without recombinant active MAPKAPK2. Peptide mass fingerprinting of trypsin-digested phosphoprotein bands using MALDI-MS and protein database analysis identified eight candidate MAPKAPK2 substrates.

FIG. 2.

FIG. 2.

Identification of 14-3-3ζ as a MAPKAPK2 substrate. (A) The mass spectra showing peptide masses (in mass per charge [_m/z_] units) obtained by MALDI-time of flight MS analysis of the phosphorylated protein band at 28 kDa. Thirty-four mass spectrum peaks were present and were queried to the theoretical masses in the entire NCBI protein database by using the Mascot search engine. A maximal 150-ppm error window and one missed tryptic cleavage were allowed. The seven peptide masses that identified 14-3-3ζ are marked by arrows. (B) The peptide masses used to identify 14-3-3ζ are compared to theoretical masses of peptides from 14-3-3ζ. The peptide sequence for each mass and the peptide coverage of 14-3-3ζ are shown. There was no significant match by peptide mass fingerprinting to the 27 peptide masses listed.

FIG. 3.

FIG. 3.

14-3-3ζ interacts with MAPKAPK2. (A) Recombinant [35S]methionine-labeled MAPKAPK2 (lanes 1 and 2) and p38α MAPK (lanes 4 and 5) were incubated with GST or GST-14-3-3ζ glutathione-coupled Sepharose. Lanes 3 and 6 show the migration of [35S]MAPKAPK2 and [35S]p38α MAPK. Proteins were separated by SDS-PAGE and subjected to autoradiography. The autoradiograph shows that MAPKAPK2, but not p38α MAPK, precipitated with 14-3-3ζ. (B) Recombinant His-14-3-3ζ was incubated with GST (lane 1), GST-MAPKAPK2 (lane 2), or GST-p38α MAPK glutathione-coupled Sepharose (lane 3). Controls were GST (lane 4), GST-MAPKAPK2 (lane 5), and GST-p38α MAPK (lane 6) without recombinant 14-3-3ζ. Precipitated proteins were separated by SDS-PAGE, followed by immunoblot (IB) analysis for 14-3-3. The immunoblot shows that 14-3-3ζ precipitated with MAPKAPK2, but not p38α MAPK.

FIG. 4.

FIG. 4.

MAPKAPK2 and 14-3-3ζ interact in neutrophil lysate. (A) Neutrophil lysates (800 μg of protein) were incubated with recombinant GST (lane 1) or GST-MAPKAPK2 (lane 2) glutathione-coupled Sepharose. Controls were GST (lane 3), GST-MAPKAPK2 (lane 4), or neutrophil lysate (200 μg of protein) (lane 5). Proteins were separated by SDS-PAGE, followed by immunoblot (IB) analysis for 14-3-3. The immunoblot shows that endogenous 14-3-3 precipitated with recombinant MAPKAPK2. (B) Neutrophil lysates (800 μg of protein) were incubated with protein A-coupled Sepharose (lane 1) or protein A-coupled Sepharose and MAPKAPK2 antibody (lane 2). Controls were neutrophil lysate (200 μg of protein) (lane 3) or MAPKAPK2 antibody alone (lane 4). Proteins were separated by SDS-PAGE, followed by immunoblot analysis for 14-3-3. The immunoblot shows that 14-3-3 immunoprecipitated with MAPKAPK2 from neutrophil lysate.

FIG. 5.

FIG. 5.

In vitro MAPKAPK2 phosphorylation of 14-3-3ζ. (A) Active recombinant MAPKAPK2 was incubated with [32P]ATP and recombinant His-14-3-3ζ in the presence of a scrambled peptide, MAPKAPK2 inhibitory peptide, or without peptide. Proteins were separated by SDS-PAGE, and phosphorylation was visualized by autoradiography. The autoradiograph shows that MAPKAPK2 phosphorylates 14-3-3ζ, and 14-3-3ζ phosphorylation is inhibited by the MAPKAPK2 inhibitory peptide. (B) Active recombinant p38α MAPK was incubated with [32P]ATP and recombinant 14-3-3ζ or recombinant MAPKAPK2. Proteins were separated by SDS-PAGE, and phosphorylation was visualized by autoradiography. The autoradiograph shows that p38α MAPK phosphorylated MAPKAPK2, but not 14-3-3ζ.

FIG. 6.

FIG. 6.

14-3-3 phosphorylation in HEK293 cells and human neutrophils. (A) HEK293 cells were transfected with vector or constitutively active MAPKAPK2 (MK2-EE). After 24 h of transfection, cells were loaded with [32P]orthophosphate, and endogenous 14-3-3ζ was immunoprecipitated. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane, and phosphorylation of 14-3-3ζ was detected by autoradiography. The autoradiograph shows that transfection of active MAPKAPK2, but not vector, resulted in phosphorylation of 14-3-3ζ. Immunoblot (IB) analysis of the same nitrocellulose membrane shows that an equal amount of 14-3-3ζ was precipitated from MK2-EE- or vector-transfected cells. (B) Human neutrophils were incubated with or without fMLP for 1, 3, or 10 min. An aliquot of the 10-min fMLP lysate was treated with 0.5 U of PP2A phosphatase prior to two-dimensional separation. Neutrophils were pretreated with 3 μM SB203580 for 60 min prior to incubation with fMLP for 10 min. Proteins were separated by two-dimensional gel electrophoresis and immunoblotted (IB) for 14-3-3. The immunoblot shows that 3- and 10-min fMLP treatments, but not the 1-min fMLP treatment, resulted in a negative isoelectric shift of a portion of 14-3-3. This pI shift was reversed by both phosphatase and SB203580 treatment. These results indicate that fMLP stimulates p38 MAPK-dependent phosphorylation of 14-3-3 in neutrophils.

FIG. 7.

FIG. 7.

MAPKAPK2 phosphorylation of 14-3-3ζ at Ser-58. Recombinant active MAPKAPK2 was incubated with [32P]ATP and recombinant 14-3-3ζ-WT or 14-3-3ζ-(S58A). Proteins were separated by SDS-PAGE, and phosphorylation was visualized by autoradiography. To demonstrate equal loading of 14-3-3 proteins, gels were also stained with Coomassie blue. The autoradiograph shows that MAPKAPK2 phosphorylated 14-3-3ζ-WT, but not 14-3-3ζ-(S58A).

FIG. 8.

FIG. 8.

Role of 14-3-3ζ phosphorylation in dimerization. (A) Recombinant 14-3-3ζ-WT, recombinant 14-3-3ζ-(S58D), and recombinant 14-3-3ζ-(S58A) were separated by nondenaturing PAGE, and proteins were detected by Coomassie blue staining. 14-3-3ζ-WT and 14-3-3ζ-(S58A) migrated at a larger molecular size than 14-3-3ζ-(S58D). (B) 14-3-3ζ-WT, 14-3-3ζ-(S58D), and 14-3-3ζ-(S58A) were incubated in the presence (lanes 1, 3, and 5) and absence (lanes 2, 4, and 6) of a chemical cross-linker, DSS. Proteins were separated by SDS-PAGE and immunoblotted (IB) for 14-3-3. The immunoblot shows a substantial reduction in 60-kDa dimer formation of 14-3-3ζ-(S58D), compared to 14-3-3ζ-WT and 14-3-3ζ-(S58A). (C) To determine the distribution of phosphorylated 14-3-3ζ between monomer and dimer, His-14-3-3ζ-WT was incubated with active recombinant MAPKAPK2 and [32P]ATP for 2 h at 30°C and then cross-linked with DSS. Under these conditions, about 20% of 14-3-3ζ was phosphorylated (data not shown). Following cross-linking, the reaction mixture was denatured with Laemmli buffer, and proteins were separated bySDS-PAGE (10% polyacrylamide). Proteins were then electrophoretically transferred to nitrocellulose. Autoradiography (right panel) and immunoblot analysis for 14-3-3 (left panel) are shown. Under these conditions, approximately equal amounts of 14-3-3 appear as monomer and dimer. The autoradiograph demonstrates that there is significantly more phosphorylation of monomer than dimer. (D) To determine the effect of MAPKAPK2 phosphorylation on dimer formation, His-14-3-3ζ-WT was incubated with 40 ng of MAPKAPK2 under three conditions prior to separation by SDS-PAGE (10% polyacrylamide), transfer to nitrocellulose, and immunoblot analysis for 14-3-3. Lane 1 contains 14-3-3ζ that was incubated with MAPKAPK2 for 2 h, but was not cross-linked with DSS, prior to SDS-PAGE. Lane 2 contains 14-3-3ζ that was phosphorylated by MAPKAPK2 for 2 h and then cross-linked with DSS prior to SDS-PAGE. Lane 3 contains 14-3-3ζ that was cross-linked with DSS prior to incubation with MAPKAPK2 for 2 h. The immunoblot shows that in the absence of cross-linking, the majority of 14-3-3ζ exists as monomer. Cross-linking after phosphorylation by MAPKAPK2 (lane 2) resulted in an equal distribution of monomer and dimer. Cross-linking before phosphorylation by MAPKAPK2 (lane 3) resulted in the majority of 14-3-3ζ migrating as dimer. Thus, phosphorylation by MAPKAPK2 resulted in a shift in 14-3-3ζ from dimer to monomer.

FIG. 9.

FIG. 9.

The S58D mutation in 14-3-3ζ impairs Raf binding. U2OS cell lysates were incubated with bead-immobilized MBP fusion proteins of wild-type 14-3-3ζ, the S58D mutant, or MBP alone. Bound proteins were analyzed by SDS-PAGE followed by immunoblotting (IB) for Raf (upper panel). An aliquot of the initial lysate is shown in the first lane. Coomassie staining (lower panel) verified that the beads contained equivalent amounts of protein.

FIG. 10.

FIG. 10.

Computational modeling of 14-3-3 dimerization. Residual surface potential for the wild-type unmodified 14-3-3ζ homodimer (A), the heterodimer of S58D mutant and wild-type unmodified 14-3-3ζ (B), the heterodimers of pSer58 and wild-type 14-3-3ζ (C), the homodimer of 14-3-3ζ-S58D mutant (D), and the homodimer of Ser-58-phosphorylated 14-3-3ζ (E) plotted at the surface of a monomer. All plots show the chain treated as ligand, with the unsatisfied residual potential color scale ranging from deep red (−40 kT/e) to deep blue (+40 kT/e).

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