Guillermo Covelo

PROTHYMOSIN ALPHA BINDS HISTONES IN VITRO AND SHOWS ACTIVITY IN NUCLEOSOME ASSEMBLY ASSAY

Prothymosin alpha (Pro Talpha) is a polypeptide which appears to be involved in cell proliferation, though its precise function has yet to be identified. Here, we report experiments which show that calf Pro Talpha selectively binds to core histones and histone H1 in vitro. Characterization of these interactions by various procedures (including affinity chromatography on Pro T alpha-Sepharose columns, immunoblotting assay and investigation of the behaviour of mixtures of Pro T alpha and histones in solution) indicated that Pro T alpha has higher affinity for core histones (particularly H3 and H4) than for H1. Similarities between the histone-binding patterns of Pro T alpha and of poly(glutamic acid) suggest that the observed histone-binding capacity resides largely in the acidic central region of Pro T alpha. However, all five histones were also bound by T alpha 1 (a peptide corresponding to the first 28 amino acids of Pro T alpha); histone binding by the N-terminal region of Pro T alpha thus cannot be ruled out. Phosphorylation of Pro T alpha does not appear to affect these interactions. In accordance with the observed capacity for histone binding, Pro T alpha (in conjunction with ATP and some Pro T alpha-binding factor/s in a thymocyte extract) was able to induce in vitro nucleosome assembly. We discuss the possibility that Pro T alpha plays a role in chromatin remodelling.

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 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.

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.