Prothymosin α Stimulates Ca2+-dependent Phosphorylation of Elongation Factor 2 in Cellular Extracts*
- From the Departamento de Fisiologı́a, Facultad de Veterinaria, Lugo and §Laboratorios de Neurociencia “Ramón Domı́nguez,” Departamento de Fisiologı́a, Facultad de Medicina and Unidad Medicina Molecular, INGO, 15705 Santiago de Compostela, Spain and ¶Ludwig Institute for Cancer Research, 75124 Uppsala, Sweden
Abstract
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 M r 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.
Footnotes
-
↵* This work was supported in part by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social and Consellerı́a de Sanidade e Servicios Sociais, Dirección Xeral de Saúde Pública, Programa de Screening de Cancer de Mama (to F. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ These two authors equally contributed to this work.
-
↵‖ To whom correspondence should be addressed: Dept. de Fisiologı́a, Facultad de Medicina, Universidad de Santiago, 15705 Santiago, Spain. Tel.: 34 81 582658; Fax: 34 81 574145; E-mail:fsfedopu{at}usc.es.
-
↵1 The abbreviations used are: PTA, prothymosin α; CaM-kinase, calcium/calmodulin-dependent protein kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; EF-2, eukaryotic elongation factor 2; W-7, N(6- aminohexyl)-5-chloro-1-naphtalene-sulfonamide.
-
↵2 A. Vidal, G. Barisone, F. V. Vega, V. Hellman, C. Wernstedt, and F. Domínquez, unpublished observations.
-
- Received November 20, 1997.
- Revision received February 16, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
