FIG. 4.

Phosphorylation of Ser78 in eEF2 kinase is sensitive to amino acid withdrawal. (A) CHO.K1 cells (90% confluent) were starved of serum for 16 h, preincubated for 1 h in serum-free Ham's F12 medium, Dulbecco's phosphate-buffered saline (D-PBS) containing 1 g of glucose per liter, or the latter containing 1 g of d-glucose per liter plus amino acids (AA) prior to treatment with insulin for 30 min as indicated. Where used, CHX was added 30 min prior to insulin stimulation. eEF2 kinase was immunoprecipitated from 100 μg of lysate protein or 20 μg of cell lysate protein and subjected to SDS-PAGE followed by Western blotting for phosphorylated and total proteins as indicated. (B) The method was the same as in panel A except that KB cells were used and were starved of amino acids for 4 h in Earle's balanced salts solution (EBSS); no treatments with CHX were included. Samples were analyzed as in panel A, except that Western blotting was also performed for phosphorylated rpS6 (Ser235) and for phosphorylation of Ser366 and Ser359 in eEF2 kinase. WB, Western blotting; IP, immunoprecipitation; eEF2k, eEF2 kinase.