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, 286 (8), 6470-8

Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway

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Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway

Paul Déléris et al. J Biol Chem.

Abstract

Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.

Figures

FIGURE 1.
FIGURE 1.
Purification of ERK3 Ser-189 kinase. A, ERK3 Ser-189 kinase assay. Purified recombinant His6-ERK3(1–365) wild type (WT), kinase-dead (KA), or nonphosphorylatable S189A (SA) mutant was incubated for 45 min at 30 °C with a lysate of HEK 293 cells. Ser-189 phosphorylation was analyzed by immunoblotting with anti-phospho-ERK3(Ser-189) and the indicated antibodies. B, strategy for the purification of ERK3 Ser-189 kinase. C, anion exchange chromatography. HEK 293 cell lysate was fractionated on a Mono Q column. Each individual fraction was assayed for Ser-189 kinase activity. D, fractions Q3 was from the Mono Q column were fractionated by gel filtration chromatography and assayed as in C. E and F, fraction GF1 from the Sephadex column incubated with His-ERK3(1–365)KA-GST protein immobilized on glutathione-agarose beads. Bound proteins were further purified by SDS-PAGE and subjected to LC-MS/MS analysis. Protein kinases with the highest Mascot scores are presented in the table.
FIGURE 2.
FIGURE 2.
Group I PAKs phosphorylate ERK3 and ERK4 in the activation loop in vitro and in vivo. A, purified recombinant catalytically inactive His6-ERK3KA or His6-ERK3SA was incubated in kinase assay buffer with recombinant wild-type (WT) or kinase-dead (KD) His6-PAK2 and [γ-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. The Coomassie-stained gel is shown at the bottom. B, recombinant wild type His6-PAK2 was incubated with His6-ERK3(1–365) WT, KA, or SA mutant, or with His6-ERK3(365–721) C-terminal domain (CTD) GST fusion in the presence of [γ-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. C and D, panels are the same as in A except that [γ-32P]ATP was omitted from the reaction. The phosphorylation of recombinant ERK3 and ERK4 fusion proteins was analyzed by immunoblotting (WB) using a phospho-ERK3(Ser-189)/phospho-ERK4(Ser-186) antibody. A duplicate gel was stained with Coomassie Blue to control for protein loading. E–G, HEK 293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed and analyzed by immunoblotting with anti-phospho-ERK3(Ser-189) and the indicated antibodies.
FIGURE 3.
FIGURE 3.
Requirement of group I PAKs for Rac1-induced activation loop phosphorylation of ERK3. A, HEK 293 cells were transfected with the indicated constructs in combination or not with SMARTpool siRNAs targeting group I PAKs. After 36 h, the cells were lysed and analyzed by immunoblotting with anti-phospho-ERK3(Ser-189) and the indicated antibodies. B, the ratio of Ser(P)-189 ERK3 to total ERK3 was determined by quantifying the anti-phospho-ERK3(Ser-189) and anti-Myc immunoblots by densitometry. The bar graph represents the mean ± S.E. (error bars) of five independent experiments as shown in A.
FIGURE 4.
FIGURE 4.
Group I PAKs phosphorylate endogenous ERK3. A, HEK 293 cells were transfected with the indicated constructs in combination or not with SMARTpool siRNAs targeting group I PAKs. After 36 h, ERK3 was immunoprecipitated from cell extracts and analyzed by immunoblotting with anti-phospho-ERK3(Ser-189) and the indicated antibodies. B, ratio of Ser(P)-189 ERK3 to total ERK3 was determined by quantifying the anti-phospho-ERK3(Ser-189) and anti-ERK3 immunoblots by densitometry. The bar graph represents the mean ± S.E. (error bars) of three independent experiments as shown in A.
FIGURE 5.
FIGURE 5.
PAKs stimulate the phosphotransferase activity of ERK3 and ERK4. HEK 293 cells were transfected with the indicated constructs in combination or not with SMARTpool siRNAs to PAK1/2/3. After 36 h, the cells were lysed, and Myc6-ERK3 WT or SA (A) or FLAG-ERK4 (B) was immunoprecipitated with anti-Myc or anti FLAG-antibody, respectively. Phosphotransferase activity was measured in a coupled kinase assay by incubating immunoprecipitated ERK3 or ERK4 with recombinant His6-MK5 and Hsp27 proteins in the presence of [γ-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel). Ponceau staining was used to control for equal His6-MK5 and Hsp27 protein loading. The activation loop phosphorylation of ERK3/ERK4 and the expression of ERK3/ERK4, Rac1CA, and PAKs were analyzed by immunoblotting of cell lysates. The bar graph represents the mean ± S.E. (error bars) of three independent experiments.
FIGURE 6.
FIGURE 6.
Rac1-PAK signaling induces MK5 phosphorylation and activation. HEK 293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed, and FLAG-MK5 was immunoprecipitated (IP) with anti-FLAG M2 antibody coupled to agarose beads. A, following elution, proteins were trypsin-digested, and the relative abundance of MK5 Thr-182 phosphorylation was analyzed by LC-MS/MS. MK5 elution and expression of the transfected constructs were monitored by immunoblot analysis (Blot) with the indicated antibodies. The bar graph represents the mean ± S.E. (error bars) of two independent experiments. B and C, MK5 kinase activity was assayed by incubating immunoprecipitated MK5 with recombinant Hsp27 protein in the presence of [γ-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel). The Coomassie-stained gel is shown at the bottom. The phosphorylation of ERK3/ERK4 and the expression of transfected proteins were analyzed by immunoblotting. The bar graph represents the mean ± S.E. (error bars) of three independent experiments.
FIGURE 7.
FIGURE 7.
Matching of ERK3 activation loop sequence to PAK1 and PAK2 consensus phosphorylation motifs. The sequence of ERK3 activation loop was matched to the position-specific scoring matrices documented by Rennefahrt et al. (16) for optimal in vitro phosphorylation by PAK1 and PAK2. Green color corresponds to residues favoring phosphorylation, whereas red color indicates unfavorable residues. Numbers refer to the order of preference.

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