Primary structure of rat thymus prothymosin alpha.

Haritos, A.A.; Blacher, Russell; Stein, Stanley J.; Caldarella, Janet; Horecker, B.L.

doi:10.1073/pnas.82.2.343

Cited by 89 publications

(61 citation statements)

References 23 publications

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“…The difficulties stem from three attributes: (a) Prothymosin ␣ is extremely acidic, with a net negative charge of ‫ف‬ 40 units (Eschenfeldt and Berger 1986;Goodall et al 1986;Frangou-Lazaridis et al 1988;Panneerselvam et al 1988;Schmidt and Werner 1991). The large number of contiguous acidic amino acids in a protein of only ‫ف‬ 12 kD (Haritos et al 1985), an unfolded conformation (Gast et al 1995), and an abundance approaching that of histone cores (Palvimo and Linnala-Kankkunen 1990;Sburlati et al 1990Sburlati et al ,1993 make prothymosin ␣ an easy target for basic proteins in binding studies performed in vitro. The relevance of binding data is, accordingly, problematic.…”

Section: S U M M a R Ymentioning

confidence: 99%

Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α

Enkemann

Ward

Berger

2000

J Histochem Cytochem.

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S U M M A R YProthymosin ␣ is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin ␣ appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin ␣ molecules, but not prothymosin ␣ fused to ␤ -galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin ␣ shared domains with mobile proteins such as Ran, transportin, and karyopherin ␤ , which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin ␣ . The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin ␣ is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus. T he function of prothymosin ␣ remains unclear despite many attempts to determine both its binding partners and its cellular location. Although the levels of the protein and its mRNA unquestionably increase in proliferating mammalian cells (Eschenfeldt and Berger 1986;Gómez-Márquez et al. 1989;Bustelo et al. 1991;Tsitsiloni et al. 1993), precisely what prothymosin ␣ does, where it does it, and what other macromolecules participate are questions with multiple unsatisfactory answers. The difficulties stem from three attributes: (a) Prothymosin ␣ is extremely acidic, with a net negative charge of ‫ف‬ 40 units (Eschenfeldt and Berger 1986;Goodall et al. 1986;Frangou-Lazaridis et al. 1988;Panneerselvam et al. 1988;Schmidt and Werner 1991). The large number of contiguous acidic amino acids in a protein of only ‫ف‬ 12 kD (Haritos et al. 1985), an unfolded conformation (Gast et al. 1995), and an abundance approaching that of histone cores (Palvimo and Linnala-Kankkunen 1990;Sburlati et al. 1990Sburlati et al. ,1993 make prothymosin ␣ an easy target for basic proteins in binding studies performed in vitro. The relevance of binding data is, accordingly, problematic. (b) Antibodies raised to prothymosin ␣ or peptides derived from it are invariably low in titer and broad in specificity. Such antibodies, particularly those directed against the N-terminus, crossreact with materials from crabs, insects, protozoans, fungi, and bacteria (Oates and Erdos 1989). However, the fully sequenced yeast genome does not code for prothymosin ␣ or related proteins, and homologous proteins in bacteria and homologous genes in a broad swath of the evolutionary spectrum up to and including reptiles h...

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Section: S U M M a R Ymentioning

confidence: 99%

Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α

Enkemann

Ward

Berger

2000

J Histochem Cytochem.

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“…The protein is neither a precursor for processed polypeptides nor specifically associated with the thymus nor a member of a family with ␤ or ␥ homologues (1)(2)(3). Instead, it is probably the most acidic naturally occurring polypeptide in the eukaryotic world, with 54 carboxyl groups in 109 amino acids, resulting in an isoelectric point at or below pH 3.5 (1,2,4). The mRNA for prothymosin ␣ is distributed ubiquitously among mammalian nucleated cells and tissues (1).…”

mentioning

confidence: 99%

Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

Trumbore

Wang

Enkemann

et al. 1997

Journal of Biological Chemistry

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2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [ 32 P]prothymosin ␣ or a [ 32 P]histidine-tagged variant resembled the pH stability curve of acetyl phosphate. 3) After a brief incubation at pH 7, these 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 that most of prothymosin ␣'s phosphates are subject instantaneously to hydrolysis, based on the observation that greater than 90% of the phosphate initially found at pH 7 disappeared at the extremes of pH. Rapid loss of phosphate was not affected by the presence of phosphatase inhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertained, but the trifling amount recovered on Ser or Thr depended heavily on conditions favoring the transient survival of labile phosphate. Further analysis using COS cells lysed in the presence of sodium borohydride showed that: 1) phosphate recovered on prothymosin ␣ decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4 -8 glutamic acid residues/molecule to vanish; 3) using [ 3 H]NaBH 4 , label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [ 3 H]proline was localized almost exclusively to a peptide with pronounced homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis. Our data demonstrate that prothymosin ␣ is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.Prothymosin ␣ is a highly unusual protein with an unfortunate name. The protein is neither a precursor for processed polypeptides nor specifically associated with the thymus nor a member of a family with ␤ or ␥ homologues (1-3). Instead, it is probably the most acidic naturally occurring polypeptide in the eukaryotic world, with 54 carboxyl groups in 109 amino acids, resulting in an isoelectric point at or below pH 3.5 (1, 2, 4). The mRNA for prothymosin ␣ is distributed ubiquitously among mammalian nucleated cells and tissues (1). The protein possesses a potent nuclear localization signal and is present in amounts equivalent to those of histone H1 (5, 6). Because the amount of prothymosin ␣ mRNA (and presumably protein) found in a cell is directly proportional to cell growth, the protein is believed to play a role in cell proliferation (1). This idea was reinforced by the observation that synchronized human myeloma cells, in the presence of antisense oligodeoxyribonucleotides directed at prothymosin ␣ mRNA, were unable to divide while detectable amounts of the antisense oligonucleotides remained inside the cell (7). There are now many examples of a link between prothymosin ␣ and growth in systems as diverse as developing mouse embryos (8); normal, mitogenstimulated, and malignant lymphocytes (9, 10); and regenerating liver (10). There are also positive ...

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“…ProT~ is a highly acidic 12.5 kDa polypeptide originally isolated from rat thymus [1,2] and subsequently detected in a wide range of organisms and tissues [3][4][5]. There is a high degree of homology among the ProT~ and ProTa cDNAs of different species [6 11].…”

Section: Introductionmentioning

confidence: 99%

The prothymosin α gene is specifically expressed in ectodermal and mesodermal regions during early postimplantation mouse embryogenesis

Amo¹,

Freire²

1995

FEBS Letters

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Prothymosin a (ProTa) is a highly acidic nuclear protein, once believed to have an extracellular immunoregulatory role but more recently implicated in cell proliferation and/or differentiation. Several recent studies have revealed that ProTa mRNA is present during embryogenesis. However, these studies did not investigate the spatial distribution of ProTa mRNA in the embryo. Here we present a detailed study of the spatial distribution of ProTa mRNA during the early stages of postimplantation development (6.5--12.5 dpc) of the mouse. Three findings are of particular interest. First, ProTa mRNA levels increase during the early postimplantation stages (6.5-8.5 dpc) of mouse embryogenesis. Second, ProTcx mRNA is not uniformly distributed in the mouse embryo, but is present in a spatially specific manner. Third, we have observed that the mouse ProTa gene is expressed almost exclusively in ectodermal and mesoderm-derived structures, and not in cells which give rise to the definitive endoderm.

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Primary structure of rat thymus prothymosin alpha.

Cited by 89 publications

References 23 publications

Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α

Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α

Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

The prothymosin α gene is specifically expressed in ectodermal and mesodermal regions during early postimplantation mouse embryogenesis

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