Identification and characterization of translational elongation factor-1alpha as a substrate of receptor tyrosine kinase Nyk

Phosphotyrosine (PY)-independent interactions between RTKs and their substrates was revealed as an alternative mechanism of RTK-mediated signaling. In an effort to further characterize Nyk-mediated signal transduction, we have used the cytoplasmic domain of a kinase-deficient mutant of Nyk as bait in the yeast two-hybrid system to identify proteins that interact with Nyk in PY-independent manners. Seven interacting clones were found to be true positives after reconstitution experiments. Interestingly, four of them encode two homologous GTP-binding proteins, eEF-1A (translational elongation factor 1α) and GSPT1 (G1 to S phase transition).; I have further confirmed the PY-independent interaction between Nyk and eEF-1A by immunochemical experiments. First, overexpressed Nyk kinase-active or kinase-deficient mutants coimmunoprecipitated with overexpressed or endogenous eEF-1A protein. Second, endogenous eEF-1A associates with the chimeric Fms-Nyk receptor in a PY-independent manner. Third, endogenously-expressed Nyk and eEF-1A coimmunoprecipitated with each other without activating the Nyk receptor. By using a series of eEF-1A deletion mutants, the Nyk-interacting region was more precisely mapped to residues 309–412 located at the C-terminal end of eEF-1A.; eEF-1A is a highly abundant, multifunctional protein. Post-translational modification was shown as an important mechanism in regulating its biological functions. The data presented here have demonstrated that eEF-1A is a substrate of the Nyk receptor tyrosine kinase. My results have shown that overexpressed eEF-1A is tyrosine-phosphorylated only when it is coexpressed with kinase-active, but not kinase-deficient Nyk. In addition, using Fms-Nyk/NIH3T3 cells as an alternative way of activating Nyk kinase activity, endogenous eEF-1A is tyrosine-phosphorylated when cells are treated with the Fms receptor ligand, CSF-1. In vitro kinase experiments further confirmed that Nyk specifically phosphorylated eEF-1A protein on tyrosine residues. One of the major phosphorylation sites was mapped to tyrosine residue 418, although other phosphorylation sites are also apparent.; My results demonstrate that eEF-1A can be physically associated with Nyk independent of receptor phosphorylation. After receptor activation, eEF-1A is tyrosine-phosphorylated by Nyk. This post-translational modification may be involved in modulating the biological activities of eEF-1A as well as transmitting Nyk-mediated signals.