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...
Translation is down‐regulated in response to a variety of stresses. Under moderate stress, p21‐activated protein kinase (Pak2) is activated by Cdc42 and induces cytostasis, while caspase 3 cleaves and activates Pak2 under apoptotic conditions. Pak2 inhibits translation through phosphorylation of initiation factor 4G and the serine/threonine kinase Mnk1. The present study shows that Pak2 phosphorylates three ribosomal proteins, S6 and S10 in the small ribosomal subunit and L34 in the large ribosomal subunit. S10 and L34 are phosphorylated by Cdc42‐activated and caspase‐cleaved Pak2, while S6 is phosphorylated only by caspase‐cleaved Pak2 at one site on serine 235. The same site is phosphorylated during early apoptosis following treatment of 293T cells with H2O2. Addition of active Pak2 to a reticulocyte lysate results in the phosphorylation of S6 and S10, inhibiting protein synthesis. Reconstitution of reticulocyte lysate with ribosomes phosphorylated by caspase‐activated Pak2 in vitro, inhibits translation by 33%, as compared with lysate reconstituted with non‐phosphorylated ribosomes. Active and inactive Pak2 binds to ribosomes and 40S ribosomal subunits, but not to 60S subunits. In summary, Pak2 is shown to directly down‐regulate translation through phosphorylation of ribosomal proteins.
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