PROTHYMOSIN-ALPHA IN-VIVO CONTAINS PHOSPHORYLATED GLUTAMIC-ACID RESIDUES

Human and monkey prothymosin alpha contain activated carbonyl groups o n glutamic acid residues. Three lines of evidence indicate the existen ce of unusual phosphates. 1) Prothymosin alpha continued to be metabol ically labeled with [P-32]orthophosphoric acid despite a mutation at S er(1), the sole site of phosphate in purified bovine prothymosin alpha (Sburlati, A. R., De La Rosa, A., Batey D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L . (1993) Biochemistry 32, 4587-4596), 2) Immediately upon cell lysis, the pH stability curves of metabolically label ed native [P-32]prothym osin alpha or a [P-32]histidine-tagged variant resembled the pH stabil ity curve of acetyl phosphate. 3) After a brief incubation at pH ?, th ese curves changed from a pattern diagnostic for an acyl phosphate to that characteristic of a serine or threonine phosphate, an observation consistent with transfer of phosphate in vitro. Our data indicate tha t most of prothymosin alpha's phosphates are subject instantaneously t o hydrolysis, based on the observation that greater than 90% of the ph osphate initially found at pH 7 disappeared at the extremes of pH. Rap id loss of phosphate was not affected by the presence of phosphatase i nhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertain ed, but the trifling amount recovered on Ser or Thr depended heavily o n conditions favoring the transient survival of labile phosphate. Furt her analysis using COS cells lysed in the presence of sodium borohydri de showed that: 1) phosphate recovered on prothymosin alpha decreased 8-fold when lysates were treated with borohydride; 2) the reagent caus ed 4-8 glutamic acid residues/molecule to vanish; 3) using [H-3]NaBH4, label was introduced into proline, a product derived from reductive c leavage of phosphoglutamate; and 4) [H-3]proline was localized almost exclusively to a peptide with pronounced homology to the histone bindi ng site of nucleoplasmin, a chromatin remodeling protein found in Xeno pus laevis. Our data demonstrate that prothymosin a is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.