Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.     

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.     

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.     

Tissue-specific and differential expression of prothymosin alpha gene during rat development.

We have analyzed the RNA expression of prothymosin alpha (ProT alpha) gene during rat development in several tissues and compared it to that of two proteins related to cell proliferation: proliferating cell nuclear antigen (PCNA)/cyclin and histone H3 (H3). The expression of ProT alpha gene was found to be regulated in a developmental and tissue-specific manner. The mRNA levels of ProT alpha followed a similar time-course in liver, brain, kidney, and testis, being highly increased in the early periods of postnatal development. However, in thymus ProT alpha mRNA showed only moderate changes throughout development. Our findings suggest that ProT alpha participates in developmental processes like cell proliferation and/or differentiation.     

Prothymosin alpha, a mammalian c-myc-regulated acidic nuclear protein, provokes the decondensation of human chromosomes in vitro

Prothymosin (ProT alpha) is an acidic nuclear protein, widely distributed in mammalian cells, whose expression is regulated by c-myc and linked to cell proliferation. ProT alpha interacts with histone H1 via its acidic domain, and its overexpression provokes the unfolding of chromatin fibers. Here we show that incubation of human native metaphase chromosomes with ProT alpha induces their extensive unravelling suggesting a function of this protein in chromosome decondensation.     

Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor.

A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (approximately 49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-1 gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-1 genes establish a new family encoding multiple membrane-spanning proteins.     

Prothymosin alpha expression in lymph nodes and tonsils: an optical and ultrastructural study.

Prothymosin alpha (ProT alpha) distribution in human and rat lymph nodes and human tonsils was studied by means of immunohistochemical methods, using specific antibodies raised against thymosin alpha 1. We observed ProT alpha immunoreactivity in lymphoid cells of the germinal centers both in humans and rats. In human tonsils, positive cells were also seen in the basal layer of the surface epithelium. These results support the hypothesis that ProT alpha expression is restricted to actively proliferating cells. Immunoelectron microscopy revealed that ProT alpha was located in the nucleus, mainly in the border between euchromatin and heterochromatin.     

Cell-cycle-dependent expression of human ornithine decarboxylase

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.     

Prothymosin alpha

Prothymosin alpha (ProT alpha) is a highly acidic protein widely distributed in mammalian cells. Since its discovery in 1984, the biological role of this protein has been controversial. Initially, ProT alpha was considered a thymic factor with a hormonal-like role in the maturation of T-lymphocytes. However, molecular and cellular analyses led to conclude that ProT alpha is a nuclear protein required in proliferation events while failing to show a clear immunological effect. The involvement of ProT alpha in changes in the compaction state of chromatin has been recently elucidated with the demonstration that this protein induces the unfolding of chromatin fibres in a process that seems to be mediated by the interaction of ProT alpha with histone H1. This finding opens up new perspectives in the study of the dynamics of the genetic material in mammalian cells. Furthermore, the relationship between ProT alpha and apoptosis as well as with proliferation makes this protein an attractive target in the search for modulators of cell death and tumour growth.     

Cell growth and gene rearrangement signals during the development of T lymphocytes within the thymus

The thymus provides signals that control the proliferation and differentiation of T lymphocytes and select the repertoire of T-cell specificities. Antibodies to CD3 molecules inhibit full rearrangement of T-cell receptor beta chain genes in organ cultures of early embryo mouse thymus. Whether this effect is mediated through gamma delta CD3 expressing cells, which are present in small numbers at this stage, or through low amounts of CD3 on alpha beta precursor cells is unclear. A requirement for special gene rearrangement signals within the thymus is supported also by the observations that growth factors such as IL-2 and IL-4, although stimulating proliferation of precursor cells removed from the thymus, do not induce full T-cell receptor gene rearrangements. Recent studies show that newly formed thymic lymphocytes expressing alpha beta CD3 receptors are targets for negative selection (deletion) as a means of removing autoreactive cells. Signalling to immature thymocytes via the alpha beta CD3 complex induces the activation of endogenous endonucleases that cleave DNA into oligonucleosomal fragments. We suggest that the activation of this mechanism is the means by which autoreactive cells are removed.     

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.     

HTLV-1 propels thymic human T cell development in "human immune system" Rag2⁻/⁻ gamma c⁻/⁻ mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ?-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2?/?γ(c)?/? mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2?/?γc?/? mice, mature single-positive (SP) CD4? and CD8? cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2?/?γ(c)?/? animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.     

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.     

Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues

Blot hybridization analyses have established the presence of mRNAs for prothymosin alpha (ProT alpha) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProT alpha and its mRNA were found in highest concentrations in spleen and thymus, followed by lung, kidney, and brain, with lowest concentrations in liver. On the other hand, liver contained highest concentrations of ParaT and the mRNA for ParaT, with lowest levels present in spleen and thymus. In comparison to tissues from young (6-8 week) mice, older (18 month) mice contained lower concentrations (20-40%) of both polypeptides, with qualitatively similar decreases in mRNA content.     

Prothymosin alpha is processed to thymosin alpha 1 and thymosin alpha 11 by a lysosomal asparaginyl endopeptidase

Thymosin alpha(1) (T alpha(1)) and thymosin T alpha(11) (T alpha(11)) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin alpha (ProT alpha), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProT alpha, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProT alpha to generate T alpha(1) and T alpha(11). In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginyl-glycine residues in the ProT alpha sequence; specifically, Asn(28)-Gly(29) and Asn(35)-Gly(36) residues are cleaved with similar efficiency in vitro to generate T alpha(1) and T alpha(11), respectively. By contrast T alpha(1) is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProT alpha. The data here reported demonstrate that T alpha(1) is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role.