Phosphorylation of Mnk1 by Caspase-activated Pak2/γ-PAK Inhibits Phosphorylation and Interaction of eIF4G with Mnk

Abstract

Within the framework of translational initiation, one of the major points of regulation involves the recruitment of the mRNA to the 43 S pre-initiation complex. Recruitment is mediated by members of the group four initiation factors (eIF4), 1 the most prominent member of which is the cap-binding complex of eIF4F. eIF4F is a heterotrimeric protein complex consisting of the 25-kDa cap-binding protein eIF4E, the 46-kDa bi-directional RNA helicase eIF4A, and the 220-kDa scaffold protein eIF4G. eIF4F (via eIF4E) binds to the m 7 G-cap of mRNAs along with eIF4B and eIF4H, positioning eIF4A to unwind mRNA secondary structures 5Ј to the AUG start codon (reviewed in Refs. 1-7). Unwinding of the secondary structural elements presumably facilitates the binding of the 43 S preinitiation complex to the eIF4⅐mRNA complex.Both eIF4E and eIF4G are phosphoproteins (reviewed in Refs. 8 and 9). While the phosphorylation of eIF4G has not been well characterized, the site in eIF4E phosphorylated in vitro and in vivo has been identified as Ser 209 (10). eIF4E is phosphorylated at this site in vitro by the mitogen-activated protein kinase-interacting kinases 1 and 2 (Mnk1 and -2) and by protein kinase C (11-15). Phosphorylation of eIF4E and eIF4G is stimulated in vivo by insulin, progesterone, tumor necrosis factor ␣, interleukin-1␤, and phorbol ester (PMA) (15)(16)(17)(18)(19)(20). eIF4G is phosphorylated in vitro by protein kinase C, multifunctional S6 kinase, and the p21-activated protein kinase Pak2/␥-PAK (11,21,22). Two gene products of eIF4G have been identified, eIF4GI and -II. Raught et al. (23) has identified three sites that are phosphorylated in response to serum within a putative hinge region (amino acids 1035-1190) in the C terminus of human eIF4GI and one site (Ser 274 ) in the N terminus. Two unidentified serum-repressed phosphorylation sites in eIF4G were also observed. Tuazon et al. (21) showed the rate of phosphorylation/dephosphorylation of eIF4E is significantly greater in the eIF4F complex than with purified eIF4E, suggesting that regulation of eIF4E by phosphorylation occurs primarily on eIF4F. Phosphorylation of eIF4F by protein kinase C or the eIF4G subunit of eIF4F by multifunctional S6 kinase stimulates translation in a reconstituted protein-synthesizing system dependent on eIF4F (22). However, overall the role of phosphorylation of 4E and 4G is not well understood.Mnk1 and -2 are activated by the MAP kinases Erk1 and -2 and p38 (18,19). Activation of Mnk occurs upon phosphorylation at two sites, Thr 197 and Thr 202 . An additional residue, Thr332 in mouse Mnk2 and Thr344 in human Mnk1, has been identified as a phosphorylation site for Erk2 (14,15,24). Ser 22 has also been shown to be phosphorylated in vitro, but the protein kinase has not been identified (24). Support for phosphorylation of eIF4E by Mnk1 and -2 in vivo comes from studies utilizing kinase-inactive and constitutively active mutants of Mnk1 (15). Kinase-inactive mutants of Mnk1 expressed in 293 cells inhibit the mitogen-induced phosphorylatio...

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

“…Expression and Purification of Recombinant Proteins-The expression and purification of two fragments of eIF4G-1 (NCBI accession number NP_886553) have been described previously; eIF4G(589 -1600) is aa residues 589 -1600 with an N-terminal tag consisting of thioredoxin, His 6 , and S-peptide (52), and eIF4G(1357-1600) is similar except with aa residues 1357-1600 (53). Recombinant human MNK1 and MNK2 were expressed from plasmids pET14-His 6 -Mnk1 and pET14-His 6 -Mnk2, respectively (54).…”

Initiation factor modifications in the preapoptotic phase

“…141 As such, the modified eIF4F complex present in apoptotic cells at early times may still be able to support either cap-dependent 110 or -independent initiation. 149 In addition, the decrease in the phosphorylation state of eIF4E during apoptosis may reflect the activation of Pak2, 140 but it could also be a consequence of eIF4G cleavage because the binding site for the eIF4E kinases, Mnk1/2, is not present in M-FAG. 148 When HeLa cells are exposed to the cytotoxic ligand TRAIL, apoptosis is rapidly induced and translation rates are severely, but not completely, inhibited.…”

“…An example of the latter is the caspase-mediated activation of the signalling molecule, Pak2, which impinges on eIF4F complex assembly. Once cleaved, this kinase has the ability to phosphorylate Mnk1 and, without influencing its kinase activity, reduce the binding of Mnk1 to eIF4GI 140 prior to cleavage of eIF4GI. Any or all of the above events could potentially contribute to the observed inhibition of protein synthesis, and it is likely that the relative importance of the various changes may be different at distinct stages of the ongoing apoptotic response.…”

Features of the Catalytic Domains and C Termini of the MAPK Signal-integrating Kinases Mnk1 and Mnk2 Determine Their Differing Activities and Regulatory Properties

“…-ERK has a conserved common docking site for its inactivators (the MKPs) and substrates such as the Mnks (2,18). One would expect that the stable binding of (P)ERK to Mnk2 could protect it from dephosphorylation, by preventing it from binding to MKPs.…”

“…Recently it has been reported that phosphorylation of Mnk1 by caspase-activated Pak2/␥-PAK inhibits the phosphorylation of Mnk1 and its interaction with eIF4G (18), but the basis of this effect was not clear. Also, previous data showed that the Mnks dissociate from eIF4G after TPA stimulation (3).…”