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

Abstract

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...

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

“…On the other hand, monoclonal antibodies are usually characterized by rather low affinity for the corresponding antigen, and consequently, they are also difficult to apply to the development of sensitive competitive immunoassays. This is probably true for the few monoclonals for ProTa reported until now in the literature (Staehli et al 1983;Sukhacheva et al 2002); as a consequence, few immunoassays have been reported for a thymosins and even fewer have been applied to the analysis of biological samples (Panneerselvam et al 1987;Tsitsiloni et al 1994;Costopoulou et al 1998;Mitani et al 2000), whereas their quantitative experimental results seem to vary and therefore have been approached with caution by some researchers (Enkemann et al 2000b). However, provided that their specificity is appropriate, carefully selected antibodies for ProTa may be of great value for certain clinical applications and especially for the immunostaining of tissue samples, in which they can be used in excess.…”

“…We strongly believe that the ProTa molecule does behave in a tricky way in the Western blot (Freire et al 2002;Lal et al 2005), and the slightest changes in the experimental conditions may greatly affect its immobilization onto the membrane, which is probably the main reason for the often reported poor Western blotting results. However, under Enkemann et al (2000b), who used COS-1 and NIH3T3 cells transfected with genes of epitope-tagged ProTa proteins in combination with antibodies against the epitope tags. The intensity of the fluorescent signal was stronger in the cancer cells than in normal ones.…”

Overexpression of the Oncoprotein Prothymosin α Triggers a p53 Response that Involves p53 Acetylation

“…The use of cell lines with different genetic alterations are likely to be important, and cell lines with an impaired p53 pathway may predominantly proliferate in response to PTMA. It is furthermore very likely that protein tags significantly alter results for PTMA, such as subcellular localization and biological functions (52,53). We have tested NH 2 -terminally tagged PTMA (FLAG, Myc, and glutathione S-transferase tags) in p53 reporter gene assays and found that none were able to stimulate p53 activity, despite expression levels similar to native PTMA.…”

Estrogen Regulation of Prothymosin Alpha

“…For these studies we used immunofluorescence staining to determine if the same cells that express PTa are also proliferating. PTa has been proposed to move between nuclear and cytoplasmic compartments (Enkemann et al, 2000). However the relative importance of nuclear and cytoplasmic localization on its proliferative effects has not been examined.…”