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. 2003 Aug;23(16):5692–5705. doi: 10.1128/MCB.23.16.5692-5705.2003

FIG. 2.

FIG. 2.

Mnk2a and Mnk2b can both phosphorylate eIF4E. (A) Amino acid sequences of residues 50 to 75 of human Mnk2a and Mnk2b. The basic region is boldfaced. The deletion of the whole region (in ΔBR) and the triple Ala mutations made in this study (KKR/AAA and KKK/AAA) are underlined. (B) The basic region is involved in eIF4G binding. HEK293 cells were transfected with the indicated constructs (the empty vector is pCS3MT), and after harvesting of the cells, Myc-tagged proteins were immunoprecipitated. Aliquots of the purified proteins were analyzed directly by Western blotting to determine expression levels (lower panel), and coimmunoprecipitated eIF4G was detected with antibodies directed against the C terminus of eIF4G (22). 2a, Mnk2a; 2b, Mnk2b. Both ΔBR constructs lack the sequence KKRGKKKKR (residues 60 to 68) (upper panel). (C) Expression of Mnk2a and Mnk2b. HEK293 cells were transiently transfected with the indicated pCS3MT constructs, and the cells were harvested as described in Materials and Methods. The Myc-tagged proteins were immunoprecipitated with anti-Myc antibodies, and the levels of expression were analyzed by Western blotting. (D) The immunoprecipitated kinases, as shown in panel B, were tested in vitro in the presence of [γ-32P]ATP for their abilities to phosphorylate recombinant eIF4E as described in Materials and Methods. The part of the autoradiogram showing labeled eIF4E is shown. (E) In vivo activitiesof human Mnk2a and human Mnk2b were assessed by purification of the endogenous eIF4E from extracts of the transfected cells and separation of the phosphorylated (eIF4E+P) and nonphosphorylated (eIF4E) forms by one-dimensional isoelectric focusing. (F) Coimmunoprecipitation of ERK was detected by probing the blot used in panel A with ERK-specific antibodies. (G) Binding of Mnk2a to eIF4G enhances eIF4E phosphorylation. HEK293 cells were transfected with an empty vector (pCS3MT) or vectors encoding Myc-tagged wild-type Mnk2a or Mnk2a with the basic region deleted. After harvesting of the cells, the phosphorylation state of endogenous eIF4E was analyzed by isoelectric focusing as described in Materials and Methods. Annotation is as for panel E.