Concepción Sarandeses

Identification of nuclear-import and cell-cycle regulatory proteins that bind to prothymosin α

Prothymosin alpha (ProT alpha) is a nuclear protein that is widely distributed in mammalian tissues, and is thought to play a role in cell proliferation. In an attempt to shed light on this role, affinity chromatography on ProT alpha-Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes (NC37 cells) that interact with ProT alpha in vitro, and thus may interact with ProT alpha in vivo. Immunoblotting techniques were used to screen the ProT alpha-binding fractions for histones and other proteins involved in nuclear transport and cell-cycle control. The most abundant ProT alpha-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear-transport proteins, karyopherin beta1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; while tranportin, CAS, and Ran BPI were not detected. Of the cell-cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at very low concentrations; while cyclin D1, cyclin D3, Cip1, and Kip1 were not detected. These results suggest (i) that ProT alpha is transported into the nucleus by the karyopherin beta1-Rch-1 complex, and (ii) that ProT alpha may interact in the nucleus with proteins involved in DNA metabolism and cell-cycle control.

The presence and cytotoxicity of CD16+CD2− subset from PBL and NK cells in long-term IL-2 cultures enhanced by Prothymosin-α

The aim of this study was to characterize the LAK cell subpopulation on which Prothymosin-alpha (ProT alpha) exerts its enhancing effect on cytotoxicity. We investigated the role of ProT alpha on LAK induction from peripheral blood lymphocytes (PBL) and NK-enriched cells, both cultured for 5 weeks with IL-2 and ProT alpha. The different cultures were separated into several subsets throughout the culture time using two color fluorescence activated cell sorting (FACS) and CD56, CD16, CD2 and CD8 monoclonal antibodies. Each cell subset was then tested for cytotoxicity against K562 and Daudi cells. The CD16+ CD2- subset from both, PBL and NK-enriched cells, was the only subset on which ProT alpha had an effect, significantly enhancing this subpopulation. Within the CD16 population, the cells CD16+ CD2+ were the most cytotoxic, although CD16+ CD2- cells were also cytotoxic. ProT alpha only potentiated the cytotoxicity of CD16+ CD2- subset significantly, with 29% and 41% for K562 and Daudi cells, respectively.

The M2-type isoenzyme of pyruvate kinase phosphorylates prothymosin α in proliferating lymphocytes

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.

Binding of 125I-prothymosin α to lymphoblasts through the non-thymosin α1 sequence

Abstract The important immunological activities of Thymosin α1 (Tα1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin α (ProTα) is a highly acidic polypeptide, first isolated as the putative precursor of Tα1. However ProTα is now known to be more immunoreactive than Tα1 in certain in vivo and in vitro assays. Recent results indicate that ProTα may be useful to design future therapeutic interventions in cancer patients if the mechanisms underlying these effects are puzzled out. With this in mind, we radiolabeled ProTα to obtain a high specific activity and a high biological activity for 125I-ProTα. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specifity for anti-ProTα and anti-Tα1. With both tools we studied the presence of binding sites for ProTα on the surface of lymphoblast cells. We conclude that ProTα binds through the non-Tα1 sequence.

Properties of the protein kinase that phosphorylates prothymosin α

The prothymosin a kinase (ProTαK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin α (ProTα), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTαK is more effectively activated by Mn2+ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin α1 and thymosin α11, derived from the amino terminus of ProTα, despite the fact that the sites of phosphorylation of ProTα are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProTα and ProTαK activity. ProTαK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTαK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTαK, which is therefore presumably phosphorylated by another kinase.

The influence of phosphorylation of prothymosin α on its nuclear import and antiapoptotic activity

Phosphorylation of prothymosin α (ProTα) appears not to affect its influence on chromatin remodelling. To determine whether it affects nuclear import or cytosolic antiapoptotic activity, cells were transfected with vectors generating tagged recombinant ProTα (rProTα), either wild-type (rProTα-wt), which is partially phosphorylated posttranslation or the nonphosphorylatable rProTα-T7A. Immunofluorescence microscopy showed the predominant location of native ProTα, rProTα-wt, and rProTα-T7A in the nucleus. The activity of caspases 9 and 3 following apoptosis induction treatment (staurosporine) indicated reduction of apoptosis by rProTα-wt but not by rProTα-T7A. It is concluded that phosphorylation of ProTα is required for its antiapoptotic activity, but it does not affect its nuclear import.

Phosphorylation of Prothymosin α. An Approach to Its Biological Significance

Prothymosin α (ProTα), the precursor of the thymosin α1 and thymosin α11, is a 109-111 amino acids protein widely distributed in the mammalian tissues that is essential for the cell proliferation and survival through its implication on chromatin remodeling and in the proapoptotic activity. ProTα is phosphorylated at Thr residues by the M2 isoenzyme of the pyruvate kinase in a process that is dependent on the cell proliferation activity, which constitutes a novel dual functionality of this enzyme. The Thr residues phosphorylated are apparently dependent on the carcinogenic transformation of the cells. Thus, in normal lymphocytes residues Thr11 or Thr12 are phosphorylated in addition to a Thr7 residue, while in tumor cells Thr7 is the only residue phosphorylated. Phosphorylation of ProTα seems to be related to its antiapoptotic activity, although other possibilities cannot be discarded.

Prothymosin α interacts with SET, ANP32A and ANP32B and other cytoplasmic and mitochondrial proteins in proliferating cells

Prothymosin α (ProTα) is an acidic protein with a nuclear role related to the chromatin activity through its interaction with histones in mammalian cells. ProTα acts as an anti-apoptotic factor involved in the control of the apoptosome activity in the cytoplasm, however the mechanisms underlying this function are still known. ProTα shares similar biological functions with acidic nuclear-cytoplasmic shuttling proteins included in SET and ANP32 family members. Using affinity chromatography, co-immunoprecipitation and chemical cross-linking, we demonstrate that ProTα interacts with SET, ANP32A and ANP32B proteins. The study by mass spectrometry of the complexes stabilized by chemical cross-linking showed that associations of ProTα consist of six highly acidic ProTα-complexes, which corresponds to differentiated interactions of ProTα either with SET or ANP32 proteins. The presence in the ProTα-complexes of cytoplasmic proteins involved in membrane remodeling and proteins implicated in the mitochondrial permeability, seems to indicate that they could be related to a cytoplasmic-mitochondrial activity. According to the cellular function of the characterized targets of ProTα, and the evolution in the composition of the diverse ProTα-complexes when proliferation activity was reduced or apoptosis induced, leads to hypothesized that ProTα interactions might be related to the proliferation activity and control of the cell survival.