ABSTRACT
In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Biosynthesis , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Blotting, Western , Cell Cycle Proteins , Cell Line , DNA Mutational Analysis , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Humans , Isoelectric Focusing , Mass Spectrometry , Molecular Sequence Data , Mutation , Peptide Mapping , Phosphorylation , RNA, Messenger/metabolism , Ribosomes/metabolism , Sequence Homology, Amino Acid , Serine/chemistry , Sirolimus/pharmacology , Spectrometry, Fluorescence , Threonine/chemistry , TransfectionABSTRACT
A discontinuous thymopoietin-like motif, composed of the fragments 97-111 and 277-307 of the molecule, as well as of residues Arg178 and Asn163 of G-actin was found. It was established that G-actin has an immunosuppressive activity regarding the humoral immune response. This activity is probably connected to the thymopentin-like sequence RKDLY, which is present in the 277-307 fragment of G-actin. The immunomodulatory activity of a series of peptide-partial sequences of G-actin was tested using plaque-forming cells (PFC) and delayed type hypersensitivity (DTH) tests. The investigated series consisted of five peptides: RKDLY (I), RKDLYANT (II), DVDIRKDLY (III), DVDIR (NO2)KDLY (IV), DVDIRKDLYANT (V). The peptides have the immunosuppressive activity regarding the humoral and cellular immune response.
