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.
A mRNA fraction purified by preparative poly- [ has been shown to.be active in certain in vitro assays for thymic hormone function (1, 4). We have described evidence for a peptide, synthesized in a cell-free system containing mRNA isolated from the thymus gland, that may be the biosynthetic precursor ofthymosin al (5). In the present work we describe the purification of the specific mRNA and additional evidence that it encodes a 16,000-dalton peptide containing the thymosin a1 sequence. Although the conversion of this peptide to thymosin a1 remains to be demonstrated, it will, for convenience, be referred to here as the "thymosin a1 precursor. Distance from origin, cm MATERIALS AND METHODS Materials.[3H]Leucine (56.5 Ci/mmol 1/Ci = 3.7 X 10'°b ecquerels) and [3H]lysine (80.5 Ci/mmol) were purchased from New England Nuclear. Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) was from Millipore, and Staphylococcus aureus protease V8 was from Miles. Synthetic thymosin cta was provided by A. Felix of Hoffmann-La Roche. The octapeptide corresponding to residues 11-18 of the thymosin a1 sequence was prepared by digestion ofa sample ofthymosin a1 with S. aureus V8 protease and purified by high-performance liquid chromatography (HPLC). Its composition was verified by amino acid analysis. Other materials were from sources as described (5).Methods. Polysomes were isolated from fresh calf thymus glands by the following procedure, which was carried out at 40C under sterile conditions in containers that had been sterilized at 1800C for 12 hr or soaked in 0.1% diethyl pyrocarbonate for 30 min. The glands were freed of connective tissue, minced, and blended in 3 vol of 50 mM Tris-HCl buffer (pH 7.5) containing 25 mM NaCl, 5 mM MgCl2, 2% (vol/vol) Triton X-100, 0.25 M sucrose, and 0.5 mg of heparin per ml, by using a Sorvall blender at halfspeed with five 5-sec bursts. The suspension was homogenized with five strokes at high speed in a glass/Teflon FIG. 1. Preparative polyacrylamide gel electrophoresis of thymus poly(A)-containing mRNA. Gels (10 x 80 mm) were, prepared with 4% (wt/vol) acrylamide as described by Loening (8). The electrophoresis buffer was 40 mM Tris/20 mM NaOAc/2 mM EDTA adjusted to pH 7.7 with HOAc. Prior to loading the sample, the gels were electrophoresed at 5 mA per tube for 2 hr at 4°C, and the electrophoresis buffer was replaced with fresh buffer. The solution of poly(A)+RNA was diluted to a final concentration of 8-10 A280 units in 70% (vol/vol) formamide (Merck) (deionized and vacuum distilled) containing 10% (vol/vol) of the electrophoresis buffer and 5% (wt/vol) of sucrose; this was heated to 65°C for 5 min, cooled, and applied to the top of the gel. Electrophoresis was carried out at 6 mA per tube for 3.5 hr at 4°C. The gels were sliced into 2-mm segments, and the RNA was eluted electrophoretically as follows. Each slice was placed on top of a small (1 cm) column of 3.6% (wt/vol) polyacrylamide gel (preelectrophoresed as before) in a 13 x 150 mm tube with a tapered tip and w...
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.
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