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.

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

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.

Properties of the protein kinase that phosphorylates prothymosin alpha.

The prothymosin alpha kinase (ProTalphaK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin alpha (ProTalpha), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTalphaK 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 ProTalpha. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin alphaI and thymosin alphaII, derived from the amino terminus of ProTalpha, despite the fact that the sites of phosphorylation of ProTalpha are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProTalpha and ProTalphaK activity. ProTalphaK 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 ProTalphaK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTalphaK, which is therefore presumably phosphorylated by another kinase.

Binding of 125I-prothymosin alpha to lymphoblasts through the non-thymosin alpha 1 sequence.

The important immunological activities of Thymosin alpha 1 (T alpha 1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin alpha (ProT alpha) is a highly acidic polypeptide, first isolated as the putative precursor of T alpha 1. However ProT alpha is now known to be more immunoreactive than T alpha 1 in certain in vivo and in vitro assays. Recent results indicate that ProT alpha 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 ProT alpha to obtain a high specific activity and a high biological activity for 125I-ProT alpha. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specificity for anti-ProT alpha and anti-T alpha 1. With both tools we studied the presence of binding sites for ProT alpha on the surface of lymphoblast cells. We conclude that ProT alpha binds through the non-T alpha 1 sequence.

Prothymosin alpha receptors on lymphocytes.

Recent results indicate that prothymosin alpha (ProT alpha) may be useful in designing future therapeutic interventions in cancer patients and in potentiating the immune system. We described recently the presence and characteristics of two binding sites for ProT alpha on human peripheral blood mononuclear cells (PBMC). In search of a receptor upregulation, we decided to corroborate this finding on two lymphocytic populations, (PHA-activated) lymphoblasts and YT cells. The kinetics of [125I]ProT alpha binding to lymphoblasts were fast at room temperature but with YT cells were slower. Analysis of steady-state binding data identified two binding sites in lymphoblasts with an apparent equilibrium Kd of 44-75 pM and 4228-9143 sites per cell for the high-affinity receptor and 1.7-2.9 nM and 20,534-35,044 sites per cell for the low-affinity receptor. However, it identified only one site with a Kd of 265-435 pM and 8318-27,237 sites per cell in YT cells. We conclude that exists a ProT alpha receptor in the CD3+ T cell population, and this presence is regulated. After binding to cell surface, [125I]ProT alpha is internalized in a short period of time and then degraded; therefore, we conclude that the dynamics of ProT alpha receptor turnover in part determines the concentration of ProT alpha available to induce its enhancing effects.

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

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.

Interleukin-2 killer cells: in vitro evaluation of combination with prothymosin alpha.

Interleukin-2 (IL-2) is a potent inducer of lymphokine-activated killer (LAK) activity against both NK-sensitive and NK-resistant tumor cell lines. IL-2 therapy has been associated with clinical responses, but these responses have occurred only with high, toxic doses of the cytokine. Since prothymosin alpha (ProT alpha) is able to enhance the spontaneous NK activity of cells from normal donors, we studied the effect of ProT alpha on LAK activity by IL-2. The lysis of K562 and Daudi cells by effector cells cultured with IL-2 was increased time dependently when cultures also contained ProT alpha. PBLC was separated by discontinuous density centrifugation in the LGL-enriched fractions. Within the CD16+ population, all effector cell precursors were CD2+, but the effector population after IL-2 or IL-2 + ProT alpha activation also contained CD16+CD2- cells; the CD2 molecule is thus indispensable for induction of LAK activity by IL-2 or IL-2 + ProT alpha but not for the action of activated LAK cells (or for the enhancing effect of ProT alpha). Within the CD3- CD16+ LGL population, 5 micrograms/ml ProT alpha plus 50 U/ml IL-2 was able to induce p70 IL-2R expression to a similar extent to 100 U/ml of IL-2. The use of ProT alpha to enhance the induction of LAK activity by IL-2 may be able to improve immunotherapy of cancer.

Prothymosin alpha receptors on peripheral blood mononuclear cells.

125I-Labeled prothymosin alpha (ProT alpha) was used to study the presence and characteristics of receptors for ProT alpha on human peripheral blood mononuclear cells (PBMC). The kinetics of 125I-ProT alpha binding to PBMC was fast at 37 degrees C, whilst it required 50 min to reach equilibrium at 4 degrees C and room temperature. Analysis of steady state binding data by the method of Scatchard and by unlabeled ProT alpha competition experiments identified two binding sites with an apparent equilibrium dissociation constant of 216-321 pM for the high-affinity receptor and of 11.4-21.1 nM for the low-affinity one; the sites per cell ranged from 1,479 to 1,519 and from 47,547 to 56,169, respectively. The kinetically derived equilibrium dissociation constant agreed with these data and showed no interaction between receptors.

On the anomalous behaviour on gel-filtration and SDS-electrophoresis of prothymosin-alpha.

The regulator of T-cell proliferation prothymosin alpha, is a protein with a relative molecular mass of 12 KDa as calculated from its amino acid sequence. This immunoregulator exhibits anomalous behaviour on gel-filtration and SDS-PAGE electrophoresis appearing as oligomers which are 5 or 2 fold larger than the corresponding polypeptide. These results suggest that a dimeric form of prothymosin alpha is stable to dissociation by SDS and reduction by beta-mercapto ethanol.

Prothymosin alpha enhances human natural killer cell cytotoxicity: role in mediating signals for NK activity.

We have investigated the effects of prothymosin alpha (ProT alpha) on the in vitro NK activity of various human cell populations obtained by successive purification of large granular lymphocytes (LGLs) and CD16+ cells. The results of this study indicate that ProT alpha is able to enhance the spontaneous NK activity of cells from normal donors. This effect was time and dose dependent in the range 7 to 350 nM, occurred over a wide range of effector/target cell ratios, and appeared not to require the presence of accessory cells. In addition, ProT alpha exhibited additive effects with recombinant interferon-gamma (rIFN-gamma) and recombinant interleukin-2 (rIL-2). Finally our data suggest that the effect of ProT alpha enhancing the NK cell cytotoxicity appears to involve enhancement of p70 IL-2R expression and internalization of IL-2, and was independent of cell proliferation.

Prothymosin alpha enhances interleukin 2 receptor expression in normal human T-lymphocytes.

We investigated whether the enhancement, by prothymosin alpha (Pro alpha), of the phytohaemagglutinin-stimulated proliferation of human peripheral blood mononuclear cells (PBMC) is due to its affect on the number of cells expressing the interleukin 2 receptor (IL-2R) or the surface density of IL-2R on PBMC. Peripheral blood mononuclear cells were obtained from 21 donors. For both an optimal phytohaemagglutinin (PHA) concentration (H) and a 10-fold dilution (L), their responses fell in two classes, high (h) and low (l), making four dose--response situations. Pro alpha significantly increased the number and IL-2R density of cells expressing IL-2R only when the response in its absence was about half maximal, i.e. for PBMC responding well to the low PHA stimulus (group Lh) or PBMC responding poorly to the optimal stimulus (group Hl). The enhancement of IL-2R expression in group Lh by Pro alpha was dose-dependent and paralleled by increased proliferative response. It appears not to be mediated by IL-2, since it was unaffected when IL-2 production was suppressed by cyclosporin A. The early interaction of Pro alpha with lymphocytes did not require the presence of macrophages, but macrophages were necessary during lymphocyte activation for modulation of PHA-stimulated IL-2R expression to be affected. The immunoregulatory activity of Pro alpha may prove useful for improving the decreased T-cell function associated with immunodeficiency, or for restoration of normal IL-2R expression by the lymphocytes of aged individuals.

Phytohemagglutin-stimulated human T cell: prothymosin alpha as an accessory signal.

Thymic peptide factors are known to modulate proliferation of normal human lymphocytes. In this work, we studied the effect of Prothymosin alpha (Pro alpha) on PHA-stimulated PBMC and PBLC. The observed effects of Pro alpha and thymosin alpha 1 (alpha 1) on PBMC were found to depend on the degree of cell stimulation, dose, and preincubation-time. Thymosin beta 4 (beta 4) had no effect on either cell type, regardless of the degree of stimulation, which shows that beta 4 may be used as a control peptide to work in this area. Induction of lymphoproliferation also depended on the presence of macrophages. Addition of monocytes or a cell-free monocyte culture supernatant (not containing IL-2) to the PHA-stimulated PBLC cultures resulted in T cell proliferation. Although IL-1 could not restore the PHA-induced proliferative response of isolated T cells by itself, it would enhance the helper effect of Pro alpha. Moreover, a polyclonal goat anti-human IL-2R (Tac Ag) did block the proliferative response induced by combined rIL-1 and Pro alpha, suggesting that an IL-2-dependent pathway of T cell proliferation was involved.