Abstract
Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.
Highlights
Prothymosin ␣ is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells
We examined the ability of sonicated lysates of NIH3T3 cells to phosphorylate a ϳ100-kDa protein in a series of incubations extending from 1 to 20 min
We have found that Triton lysis quantitatively releases the prothymosin ␣ in a cell into the supernatant fluids [9] and that electrophoretic analysis followed by densitometry of the extracted materials from 5 ϫ 106 NIH3T3 cells resulted in 710 arbitrary units of prothymosin ␣
Summary
Vol 274, No 26, Issue of June 25, pp. 18644 –18650, 1999 Printed in U.S.A. (Received for publication, December 15, 1998, and in revised form, April 8, 1999). Since rev is involved in the export of incompletely spliced viral RNA from the nucleus, it is tempting to speculate that prothymosin ␣ interacts with a rev-like protein produced in normal cells and that its role is to assist in the export of RNA or protein from the mammalian nucleus This view of prothymosin ␣ as a facilitator of nuclear export is entirely distinct from the previous view of the protein as a histone-binding factor involved with chromatin. Vega et al surmise that prothymosin ␣ exerts its effect on eEF-2 phosphorylation at mitosis when the nuclear and cytoplasmic compartments are no longer distinct They have implicated prothymosin ␣ in the down-regulation of translation during M phase. We conclude that prothymosin ␣ has no role to play in the regulation of eEF-2 phosphorylation
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