Prothymosin alpha stimulates Ca2+-dependent phosphorylation of elongation factor 2 in cellular extracts.
Prothymosin alpha (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.
Vega
F V
FV
Departamento de Fisiología, Facultad de Veterinaria, Lugo, 15705 Santiago de Compostela, Spain.
Vidal
A
A
Hellman
U
U
Wernstedt
C
C
Domínguez
F
F
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002457:Cell Extracts; D054736:Elongation Factor 2 Kinase; D004789:Enzyme Activation; D051379:Mice; D008934:Mitogens / Q000378:metabolism; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011498:Protein Precursors / Q000494:pharmacology*; D015398:Signal Transduction; D013947:Thymosin / Q000031:analogs & derivatives* / Q000494:pharmacology
D002457:Cell Extracts; D008934:Mitogens; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011498:Protein Precursors; C040782:prothymosin alpha; D013947:Thymosin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517690:Eef2k protein, mouse; D054736:Elongation Factor 2 Kinase; D002118:Calcium