Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase.

Abstract

A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has --5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent K; values of T3, L-thyroxine, and D-T3 are 30 aM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.The thyroid hormone 3,3',5-triiodo-L-thyronine (T3) plays an essential role in maintaining fetal and neonatal development, regulating amino acid and electrolyte transport into the cell, modulating carbohydrate, protein, and lipid metabolism, and increasing the rate of oxidative phosphorylation. No single mechanism has been found to account for all the diverse actions of-thyroid hormones.Some of the thyroid hormone effects are initiated through the interaction of T3 with nuclear receptors (see reviews in refs. 1 and 2). However, some ofthe effects of T3, such as the enhancement of enzymatic activity (3,4) and cellular amino acid accumulation and ATP synthesis (5) that occur in the absence of protein synthesis, have to be accounted for by alternative mechanism(s). Therefore, extranuclear T3 binding sites that might mediate such activities have been sought. Cheng and coworkers (6) purified a cytosolic thyroid hormone-binding protein (p58) to homogeneity. The purified protein retains its T3-binding activity and specificity (6), and two monoclonal antibodies against p58 have been developed (7). p58 has an apparent molecular weight of 60,000 by gel filtration and 58,000 by SDS/PAGE. To study its cellular function and the roles it plays in T3 action, we isolated and sequenced the cDNA that encodes p58A We found that p58 is a monomer of pyruvate kinase (ATP:pyruvate 02-phosphotransferase, EC 2.7.1.40) subtype M2 and that its conversion to the tetrameric pyruvate kinase is regulated by fructose 1,6-bisphosphate (Fru-1,6-P2).

Purification and Characterization of a Cytosolic Thyroid‐Hormone‐Binding Protein (CTBP) inXenopusLiver

“…2; Chin, 1991;Oppenheimer and Samuels, 1983). In one or more of these features, the Xenopus liver CTBP differs from several cytosolic TH-binding proteins from other species and cell types described previously (Kitagawa et al, 1987;Hashizume et al, 1989a,b;Kato et al, 1989;Lennon, 1992;Yoshizato et al, 1975;Durban and Paik, 1976;Jaffe, 1978;Jaffe and Gold, 1977). It does, however, resemble in size a 58-kDa cytosolic protein from the A431 human carcinoma cell line, rat kidney and brain, and bullfrog tadpole tail (Kitagawa et al, 1987;Hashizume et al, 1989a;Kato et al, 1989;Lennon, 1992;Durban and Paik, 1976) or in its T,-binding characteristics to CTBPs from rat kidney and brain (Hashizume et al, 1989b;Lennon, 1992).…”

“…Unlike the cytosolic retinoid-binding proteins CRABP and CRBP which do not show any variability (see MorrissKay, 1992), CTBPs from different sources or studied in different laboratories, exhibit a wide range of properties (Tata et al, 1962;Hashizume et al, 1989a;Jaffe, 1978;Kato et al, 1989;Kobayashi et al, 1991;Fanjul and Farias, 1991;Yoshizato et al, 1975;Lennon, 1992). These include a size variation of 25 -200 kDa, activation by NADPH, metal ions or -SH reagents, endogenous enzyme activities and T,-binding affinities ranging over Kd 10-10-10-5 M. Since a CTBP from Xenopus cells had not previously been described, it became important to precisely characterize the properties of xCTBP in whole cytosol and to monitor these at every step of the purification procedure.…”

“…Since the first reports of a cytosolic L-thyroxine-(TJ-binding protein in rat skeletal muscle (Tata, 1958;Tata et al, 1962), multiple CTBPs with varying characteristics, depending on the species, tissue and method of characterization, have been reported (Kitagawa et al, 1987;Dillman et al, 1974;Defer et al, 1975;Lennon, 1992;Hashizume et al, 1989b;Kobayashi et al, 1991). Interestingly, one of these has been found to be homologous to a subunit of pyruvate kinase, while another CTBP-like protein, activated by NADPH, has also been described (Kato et al, 1989;Ashizawa and Cheng, 1992;Lennon, 1992;Hashizume et al, 1989b;Kobayashi et al, 1991). Not surprisingly, a variety of functions for these CTBPs have been suggested, such as a cytosolic reservoir for thyroid hormone (TH), a carrier protein for T, from cytoplasm to nucleus and a modulator of nuclear-receptor-mediated transcription (Hashizume et a]., 1989a,b; Ashizawa and Cheng, 1992) but direct evidence to support such roles is still lacking.…”

Tissue‐dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: Its role in the regulation of amphibian metamorphosis

“…Cloning of the cDNA for a Xenopus laevis CTHBP A 1.8 kb fragment of the human CTHBP (also known as PKM2) cDNA [8] was used to screen 106 clones of an amplified cDNA library made from mRNA isolated from stage 52-54 tadpole intestine. The filters were hybrid&d as above but washed 3 times at room temperature in 2 x SSC and 0.2% SDS for 10 min each and then twice at 50" C in 0.5 x SSC and 0.5% SDS for 25 min each.…”

“…A cDNA fragment of the human CTHBP gene [8] was initially used as a probe for Northern blot analysis. A single RNA of about 2 kb in size was detected in RNAs from whole tadpoles at stage 52/54 or from the intestine alone under reduced stringency conditions (not shown), indicating the presence of a homologous frog gene.…”