The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2α from phosphorylation by kinases

doi:10.1016/j.abb.2006.06.009

Archives of Biochemistry and Biophysics

Volume 452, Issue 2, 15 August 2006, Pages 138-148

https://doi.org/10.1016/j.abb.2006.06.009 Get rights and content

Abstract

Phosphorylation of the α-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2α from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2α phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the D6/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2α and eIF2γ and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.

Section snippets

Materials and methods

All chemicals used in this study were obtained from Sigma Chemicals (St. Louis, MO), Merck (Darmstadt, Germany), ICN Biomedicals, Inc. (Aurora, OH), Fisher Chemicals (Fairlawn, NJ), or Gibco-BRL (Rockville, MD). All enzymes used in this study were purchased from New England Biolabs (Beverly, MA). [³⁵S]Methionine was purchased from Amersham Corporation.

Results and discussion

Previously, we reported that the D6/2 mutant has increased protection of eIF2α phosphorylation activity during normal growth conditions [8]. We measured its effect and the effects of five double mutants D6/2 + D251A, D6/2 + D262A, D6/2 + H331A, D6/2 + E364A, and D6/2 + E459A on the levels of eIF2α(P) and total eIF2α in serum-starved cells. The results were compared with the control cells expressing wild type p67 and EGFP alone (Fig. 1A and B). Results were normalized with the total eIF2α. Wild type p67

Acknowledgment

Kent State University Research Council supports this work.

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It is therefore likely that these domains mediate interactions with various partners, and contribute to regulate the NME process. Accordingly, it has been shown that the first N-terminal polycharged Lys-rich block of MetAP2b, previously called p67 [68], stores a POEP (protection of eIF2α phosphorylation) activity [58], preventing the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) by interacting with it [69–71]. When unphosphorylated, eIF2α can bind to the ribosome and initiate translation, while its phosphorylated form cannot bind, leading to inhibition of translation initiation [58].
N-terminal protein modifications correspond to the first modifications which in principle any protein may undergo, before translation is completed by the ribosome. This class of essential modifications can have different nature or function and be catalyzed by a variety of dedicated enzymes. Here, we review the current state of the major N-terminal co-translational modifications, with a particular emphasis to their catalysts, which belong to metalloprotease and acyltransferase clans. The earliest of these modifications corresponds to the N-terminal methionine excision, an ubiquitous and essential process leading to the removal of the first methionine. N-alpha acetylation occurs also in all Kingdoms although its extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial pathway existing only in eukaryotes. Recent studies dealing on how some of these co-translational modifiers might work in close vicinity of the ribosome is starting to provide new information on when these modifications exactly take place on the elongating nascent chain and the interplay with other ribosome biogenesis factors taking in charge the nascent chains. Here a comprehensive overview of the recent advances in the field of N-terminal protein modifications is given.
Calpain cleaves methionine aminopeptidase-2 in a rat model of ischemia/reperfusion
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Dissociation of MetAP2 from phosphorylated eIF2α causes normal protein translation to decrease while production of pro-apoptotic proteins, such as ATF4, increase due to the presence of an internal ribosome entry site, allowing bypass scanning (Boyce et al., 2005; Dever et al., 1995; Schroder and Kaufman, 2005). Bypass scanning does not occur when MetAP2 is bound to eIF2α (Datta et al., 2006) and thus dissociation of MetAP2 from eIF2α, possibly through calpain proteolysis as observed in the current study, could lead to an increase in bypass scanning of pro-apoptotic proteins. In the present study we investigated MetAP2 proteolysis in a rat MCAO model, consisting of a 1 h focal ischemia followed by harvest of tissue at either 1 h or 24 h post-reperfusion.
Ischemic stroke results in multiple injurious signals within a cell including dysregulation of calcium homeostasis. Consequently, there is an increase in the enzymatic activity of the calpains, calcium dependent proteases that are thought to contribute to neuronal injury. In addition, cellular stress due to ischemia/reperfusion also triggers a decrease in protein translation through activation of the unfolded protein response (UPR). In the present study we found that methionine aminopeptidase 2 (MetAP2), a critical component of the translation initiation complex, is a calpain substrate. In vitro calpain assays demonstrated that while MetAP2 has autoproteolytic activity, calpain also produces a stable proteolytic fragment at 50 kDa using recombinant MetAP2. This 50 kDa fragment, in addition to a 57 kDa fragment was present in in vitro digestions of rat brain homogenates. Production of these fragments was inhibited by calpastatin, the endogenous and specific inhibitor of calpain. Using an in vivo middle cerebral artery occlusion (MCAO) model only the 57 kDa fragment of MetAP2 was observed. These data suggest that calpain activation in stroke may regulate MetAP2-mediated protein translation giving calpains a larger role in the cellular stress response than previously determined.
Roles of P67/MetAP2 as a tumor suppressor
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A precise balance between growth promoting signals and growth inhibitory signals plays important roles in the maintenance of healthy mammalian cells. Any deregulation of this critical balance converts normal cells into abnormal or cancerous cells. Several macromolecules are being identified and characterized that are involved in the regulation of cell signaling pathways that connect to the cell cycle and thus they play roles as tumor promoters or tumor suppressors. In situ tumor formation needs active angiogenesis, a process that generates new blood vessels from existing ones either by splitting or sprouting. Several small molecule inhibitors and proteins have been identified as inhibitors of angiogenesis. One such protein, p67/MetAP2 also known as methionine aminopeptidase 2 (MetAP2), has been shown to bind covalently to fumagillin and its derivatives that have anti-angiogenic activity. In addition to fumagillin or its derivatives, several other small molecule inhibitors of p67/MetAP2 have been recently identified and some of these drugs are in phase III trials for cancer therapy. Although molecular details of actions toward tumor suppression by these drugs are largely unknown, a significant progress has been made to understand the structure–function relationship of p67/MetAP2 and its roles in the maintenance of the levels of phosphorylation of the ∝-subunit of eukaryotic initiation factor 2 (eIF2∝) and extracellular signal-regulated kinases 1 and 2 (ERK1/2). In this article, roles of p67/MetAP2 in the suppression of cancer development are also discussed.
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Thus, eukaryotic MetAP1 contains an N-terminal zinc finger motif that was shown to be involved in association with the ribosome in yeast (Vetro and Chang, 2002). Eukaryotic MetAP2 possesses an N-terminal poly-basic and acidic repeats that were shown to inhibit phosphorylation of eukaryotic initiation factor 2α subunit and enhance the rate of global protein synthesis (Datta et al., 1989, 2001, 2003a, 2003b, 2006, 2007; Ghosh et al., 2006). Several lines of evidence suggest that MetAP1 is constitutively expressed and is more likely to be a housekeeping enzyme, whereas MetAP2 expression is subject to regulation by cell cycle status and other extracellular signals (Chatterjee et al., 1998; Gupta et al., 1997).
The fumagillin family of natural products is known to inhibit angiogenesis through irreversible inhibition of human type 2 methionine aminopeptidase (MetAP2). Recently, fumagillin and TNP-470 were reported to possess antimalarial activity in vitro, and it was hypothesized that this inhibition was mediated by interaction with the putative malarial ortholog of human MetAP2. In this report, we have overexpressed and purified to near-homogeneity PfMetAP2 from bacteria, yeast, and insect cells. Although none of the recombinant forms of PfMetAP2 exhibited enzymatic activity in existing assays, PfMetAP2 proteins expressed in both yeast and insect cells were able to bind to fumagillin in a pull-down assay. The interaction between fumagillin and analogs with PfMetAP2 was further demonstrated using a newly established mammalian three-hybrid assay incorporating a conjugate between dexamethasone and fumagillin. Unlike human (Hs)MetAP2, it was found that PfMetAP2 is bound to fumagillin noncovalently. Importantly, a new analog of fumagillin, fumarranol, was demonstrated to interact with PfMetAP2 and inhibit the growth of both chloroquine-sensitive and drug-resistant Plasmodium falciparum strains in vitro. Antiparasite activity of fumagillin and fumarranol was also demonstrated in vivo using a mouse malaria model. These findings suggest that PfMetAP2 is a viable target, and fumarranol is a promising lead compound for the development of novel antimalarial agents.
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The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2α from phosphorylation by kinases

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgment

J. Biol. Chem.

Biochimie

Biochimie

Exp. Cell Res.

Cell

Arch. Biochem. Biophys.

J. Biol. Chem.

Biol. Chem.

Biochem. Biophys. Acta

Biochim. Biophy. Acta

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell Res.

Exp. Cell Res.

Biochemistry

Biochemistry