Prothymosin α Stimulates Ca2+-dependent Phosphorylation of Elongation Factor 2 in Cellular Extracts

• Published in: The Journal of biological chemistry 1998-04, Vol.273 (17), p.10147-10152
• Main Authors: Vega, Felix V., Vidal, Anxo, Hellman, Ulf, Wernstedt, Christer, Domı́nguez, Fernando
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Cell Extracts; Elongation Factor 2 Kinase; Enzyme Activation; Mice; Mitogens - metabolism; Peptide Elongation Factor 2; Peptide Elongation Factors - metabolism; Phosphorylation; Protein Precursors - pharmacology; Signal Transduction; Thymosin - analogs & derivatives; Thymosin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.17.10147

Prothymosin α (PTA) stimulates in a dose-dependent manner the phosphorylation of a 105-kDa protein (p105) in cell extracts from different cell types. Protein sequencing and immunological analysis indicated that this protein is elongation factor 2 (EF-2). We propose that calcium/calmodulin-dependent protein kinase III is responsible for the PTA-dependent EF-2 phosphorylation based on the following lines of evidence: (a) Ca2+ is required for the effect; (b) calmodulin enhances the reaction, and calmodulin inhibitors block the phosphorylation; and (c) no phosphorylation is seen in cell extracts depleted of calmodulin-binding proteins. To obtain a strong phosphorylated EF-2 band, we found it necessary to add PTA to cytosolic extracts from synchronized dividing cells in various phases of the cell cycle except in mitosis. Since PTA is a nuclear protein everywhere in the cell cycle except in mitosis, when it is found in the cytoplasm, we hypothesize that, if PTA activates EF-2 phosphorylation in vivo, as present data suggest, its presence in the cytoplasm during mitosis could explain why EF-2 phosphorylation is mainly restricted to that phase of the cell cycle. Moreover, other bands in addition to EF-2 were phosphorylated in a calmodulin- and PTA-dependent manner, and several of them (in a range between 50 and 60 kDa) have similar Mr to those that conform to the holoenzyme calcium/calmodulin dependent protein kinase II, suggesting that PTA could have a more general function modulating the activity of various Ca2+/CaM-dependent enzymes along the cell cycle.