Phosphorylation States of Translational Initiation Factors Affect mRNA Cap Binding in Wheat † Mateen A. Khan and Dixie J. Goss* Department of Chemistry, Hunter College and the Graduate Center of the City UniVersity of New York, New York 10021 ReceiVed February 26, 2004; ReVised Manuscript ReceiVed May 6, 2004 ABSTRACT: Phosphorylation of eukaryotic translational initiation factors (eIFs) has been shown to be an important means of regulating protein synthesis. Plant initiation factors undergo phosphorylation/ dephosphorylation under a variety of stress and growth conditions. We have shown that recombinant wheat cap-binding protein, eIF(iso)4E, produced from E. coli can be phosphorylated in vitro. Phospho- rylation of eIF(iso)4E has effects on m 7 G cap-binding affinity similar to those of phosphorylation of mammalian eIF4E even though eIF(iso)4E lacks an amino acid that can be phosphorylated at the residue corresponding to Ser-209, the phosphorylation site in mammalian eIF4E. The cap-binding affinity was reduced 1.2-2.6-fold when eIF(iso)4E was phosphorylated. The in vitro phosphorylation site for wheat eIF(iso)4E was identified as Ser-207. Addition of eIF(iso)4G and eIF4B that had also been phosphorylated in vitro further reduced cap-binding affinity. Temperature-dependent studies showed that ΔH° was favorable for cap binding regardless of the phosphorylation state of the initiation factors. The entropy, however, was unfavorable (negative) except when eIF(iso)4E was phosphorylated and interacting with eIF(iso)4G. Phosphorylation may modulate not only cap-binding activity, but other functions of eukaryotic initiation factors as well. Eukaryotic mRNAs have a common cap structure [m 7 G(5′)ppp(5′)N, where N is any nucleotide] at the 5′- terminus which plays an important role in stabilizing the mRNA structure and facilitating mRNA binding to the ribosome during initiation (1). To allow the efficient transla- tion of mRNA, an interaction is required between the cap structure and eukaryotic initiation factor (eIF)4F, 1 consisting of eIF4G, eIF4E, the cap-binding protein, (2) and sometimes eIF4A depending on the preparation. In plants, a second cap- binding protein, eIF(iso)4F, is also present. eIF(iso)4F consists of eIF(iso)4E, a 28 kDa cap-binding protein, and eIF(iso)4G, an 86 kDa polypeptide (3). In mammalian cells, it has been indicated that cap-dependent translation is regulated through the interaction of eIF4E with endogenous 4E-binding proteins (4EBP1, 4EBP2, and 4EBP3) and the interrelated phosphorylation of eIF4E and 4EBPs. Thus far, eIF4E-binding proteins have not been identified in plants. eIF4G, the large subunit of eIF4F, serves as a platform for the interaction and assembly of multiple initiation factors, including eIF4E, eIF4A (whose RNA helicase and ATPase activities are required for unwinding RNA secondary struc- ture), and eIF3 (responsible for 40 S ribosomal subunit recruitment) (4-6). In eukaryotic protein biosynthesis, the binding of mRNA to the small (40 S) ribosomal subunit is the rate-limiting step and is a key target for regulation (7-11). The presence of cap structure on eukaryotic mRNA facilitates the recruitment of translational initiation factors to allow ribosome binding and initiation at the correct start site (2, 12-15). eIF4E interacts directly with the cap via its concave surface (16, 17) and forms a complex with the scaffold protein, eIF4G (18), on its convex surface. eIF4G in turn recruits other initiation factors, such as eIF3, eIF4A, and poly(A)-binding protein (PABP) to the 5′ end of the mRNA, to generate the cap-binding complex. This is believed to promote the efficient unwinding of secondary structure in the 5′-untrans- lated region (19) and the functional circularization of mRNA believed to be necessary to promote efficient translation (2, 14, 15, 20, 21). The functional interaction between the cap and poly(A) tail is repressed following serum starvation in mammalian cells but can be reversed by exposure of the cells to insulin (22). The addition of either insulin or serum promotes rapid phosphorylation of eIF4E, eIF4G, eIF4B (23-25), and the eIF4E-binding protein (i.e., 4E-BP or PHAS-I). This phos- phorylation of 4E-BP or PHAS-I results in its release from eIF4E (26, 27), and promotes the association between eIF4G and PABP (28). In plants, PABP and several of the initiation factors are known to be phosphorylated. As in yeast and sea urchin (29), plant PABP is present as multiple phosphorylated species, which are dephosphorylated by alkaline phosphatase, confirming the nature of the modification (30). Plant eIF4B † This work was supported in part by National Science Foundation Grant MCB 0076344 (to D.J.G.) and a Professional Staff Congress City University of New York faculty award (to D.J.G.). Center in Minority Institution Award RR-03037 from the National Center for Research Resources of the National Institutes of Health supports the infrastructure at Hunter College. * To whom correspondence should be addressed. Phone: (212) 772- 5383. Fax: (212) 772-5332. E-mail: dgoss@hunter.cuny.edu. 1 Abbreviations: eIF, eukaryotic initiation factor; kDa, kilodalton- (s); 4E-BP, eIF4E-binding protein; PABP, poly(A)-binding protein; TFE, trifluoroethanol; Ant-m 7 GTP, anthraniloyl 7-methylguanosine triphosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminomethane. 9092 Biochemistry 2004, 43, 9092-9097 10.1021/bi049602b CCC: $27.50 © 2004 American Chemical Society Published on Web 06/19/2004