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Science Signaling  05 Oct 2010:
Vol. 3, Issue 142, pp. ec306
DOI: 10.1126/scisignal.3142ec306

Most plasma membrane receptors signal through complex cascades, frequently involving multiple phosphorylation events. Fewer examples are known of receptors with intrinsic kinase activity directly phosphorylating the key functional protein without using complex signaling networks. Lin et al. show that in vitro the type 1 transforming growth factor–β (TGF-β) receptor (TβR1) directly phosphorylates elongation factor eEF1A1 and that this inhibits translation. They identified eEF1A1 in a screen for TβR1 substrates and identified the phosphorylated residue as Ser300, which is within the region where the amino acid–loaded transfer RNA (aa-tRNA) interacts. Western blotting with a phosphorylation-specific antibody for pSer300 eEF1A1 showed that treatment of various cell lines with TGF-β stimulated eEF1A1 Ser300 phosphorylation in a manner dependent on the presence and activity of TβR1. Analysis of various eEF1A1 mutants, either mimicking phosphorylation (Ser300Glu and Ser300Asp) or eliminating the hydroxyl group (Ser300Ala), showed that these mutants exhibited reduced aa-tRNA binding and also had an inhibitory effect in an in vitro translation assay. Addition of a glutathione S-transferase fusion protein with the TβR1 cytoplasmic domain containing a point mutation resulting in constitutive activity also inhibited in vitro translation and promoted eEF1A1 Ser300 phosphorylation. Expression of eEF1A1 or the eEF1A1 mutants in the MCF-7 breast cancer cell line showed that the mutants inhibited translation and that only cells transfected with the wild-type eEF1A1 exhibited a TGF-β–dependent decrease in translation. With several assays for cell proliferation, the authors showed that expression of the eEF1A1 mutants inhibited cell proliferation, whereas wild-type eEF1A1 increased proliferation, but these cells were sensitive to the inhibitory effect of TGF-β. Finally, examination of normal tissue and breast cancer samples showed that the tumor cells had reduced or nondetectable eEF1A1 pSer300, whereas some adjacent stromal tissue had strong staining for pSer300 phosphorylation, as did noncancerous tissue. This is consistent with an increase in TGF-β production in the cancerous area, but with the tumor cells exhibiting decreased sensitivity while the adjacent tissue shows strong TGF-β responses.

K. W. Lin, I. Yakymovych, M. Jia, M. Yakymovych, S. Souchelnytskyi, Phosphorylation of eEF1A1 at Ser300 by TβR-I results in inhibition of mRNA translation. Curr. Biol. 20, 1615–1625 (2010). [PubMed]

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