Gavin I. Welsh

Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3‐kinase (PI 3‐kinase). The ϵ‐subunit of eIF2B is phosphorylated by glycogen synthase kinase‐3 (GSK‐3), which is inactivated by insulin in a PI 3‐kinase‐dependent manner. Here we identify the phosphorylation site in eIF2Bϵ as Ser540 and show that treatment of eIF2B with GSK‐3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3‐kinase inhibitors. Insulin‐induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

GSK3: a SHAGGY frog story

Glycogen synthase kinase 3 was discovered in mammals several years ago but only recently has it become clear that this enzyme is acutely regulated by hormones such as insulin and by growth factors. In mammals, it appears to be controlled by a signalling pathway linked to phosphoinositide 3-kinase and may regulate a range of biosynthetic processes. Evidence is now accumulating that GSK3 plays a key role in the regulation of cell fate and differentiation in many eukaryotic species.

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide‐chain initiation and is acutely activated by insulin, although it is not clear how. Inhibitors of phosphatidylinositide 3‐kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3‐kinase also prevented activation of eIF2B, while a Sos‐mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase‐3 (GSK‐3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK‐3 were regulated reciprocally. Dominant negative PI 3‐kinase abolished the insulin‐induced inhibition of GSK‐3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3‐kinase and inhibition of GSK‐3.

Activation of microtubule-associated protein kinase (Erk) and p70 S6 kinase by D2 dopamine receptors

Abstract: The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule‐associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose‐dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3‐kinase (PI 3‐kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3‐kinase‐dependent and ‐independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3‐kinase.

Nerve and epidermal growth factor induce protein synthesis and eIF2B activation in PC12 cells.

The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3′-OH kinase inhibitor wortmannin showed that both NGF- and EGF-induced protein synthesis and eIF2B activation were phosphatidylinositol-3′-OH kinase dependent. The EGF-induced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding protein-rapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein. The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.

Regulation of Protein Synthesis by Insulin Through IRS-1

Most regulatory mechanisms for the overall rate of protein synthesis described to date involve the initiation phase, which is catalyzed by the initiation factors (eIF1; Merrick and Hershey 1996; Rhoads 1999). Protein synthesis occurs in a series of discrete steps. A ternary complex of eIF2•GTP•Met-tRNAi binds to a complex of the 40 S ribosomal subunit and eIF3 to form the 43 S initiation complex. The eIF4 factors plus the poly(A)-binding protein collectively recognize the 5′-terminal cap or 3′-terminal poly(A) tract of mRNA, unwind its secondary structure, and transfer it to the 43 S initiation complex, resulting in the 48 S initiation complex. Scanning for the first initiation codon in good sequence context requires ATP hydrolysis by eIF4A and the presence of eIF1 and eIF1A (Pestova et al. 1998). Then, eIF5 stimulates GTP hydrolysis by eIF2, after which the initiation factors are replaced by the 60 S subunit to form the 80 S initiation complex. The released eIF2-GDP is recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B. The first elongator aminoacyl-tRNA is brought to the A-site by eEF1, followed by synthesis of the first peptide bond and translocation catalyzed by eEF2