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Reversible histone acetylation changes the chromatin structure and can modulate gene transcription. Mammalian histone deacetylase 1 (HDAC1) is a nuclear protein that belongs to a growing family of evolutionarily conserved enzymes catalysing the removal of acetyl residues from core histones and other proteins. Previously, we have identified murine HDAC1 as a growth factor-inducible protein in murine T-cells. Here, we characterise the molecular function of mouse HDAC1 in more detail. Co-immunoprecipitation experiments with epitope-tagged HDAC1 protein reveal the association with endogenous HDAC1 enzyme. We show that HDAC1 can homo-oligomerise and that this interaction is dependent on the N-terminal HDAC association domain of the protein. Furthermore, the same HDAC1 domain is also necessary for in vitro binding of HDAC2 and HDAC3, association with RbAp48 and for catalytic activity of the enzyme. A lysine-rich sequence within the carboxy terminus of HDAC1 is crucial for nuclear localisation of the enzyme. We identify a C-terminal nuclear localisation domain, which is sufficient for the transport of HDAC1 and of reporter fusion proteins into the nucleus. Alternatively, HDAC1 can be shuttled into the nucleus by association with another HDAC1 molecule via its N-terminal HDAC association domain. Our results define two domains, which are essential for the oligomerisation and nuclear localisation of mouse HDAC1., (Copyright 2001 Academic Press.)

Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability., (Copyright 2000 Academic Press.)

The expression of murine thymidine kinase (TK) is strictly dependent on the growth state of the cell. Expressing epitope-tagged TK in LTK cells, we have previously shown that low TK enzyme levels in G0 cells are in part due to a dramatic decrease in TK protein stability. Here we report that thymidine, one of the substrates of TK, is able to counteract the growth-arrest-specific decrease of TK expression. While TK mRNA levels and TK translation rate are almost unaffected by thymidine, the TK protein half-life rose more than sixfold after addition of the nucleoside to resting cells. The effect of thymidine is reversible and is independent of its presence during the protein synthesis of TK. Dideoxythymidine, a specific inhibitor of the TK enzyme activity, also has the capacity to increase TK protein levels in G0 cells, indicating that the substrate itself exerts the stabilising effect on the TK protein.

The expression of murine thymidine kinase (TK) is highly dependent on the growth state of the cell. The enzyme is nearly undetectable in resting (G0) cells, but TK protein levels rise dramatically when serum-stimulated cells reach the G1/S boundary. To study post-transcriptional regulation of TK expression, Ltk- cells were stably transfected with the coding region of the TK cDNA under the control of a constitutive SV40 promoter. While TK mRNA levels were growth independent in this cell line, TK protein expression and enzyme activity were low in resting cells but increased strongly after growth stimulation by serum. Measurements of translation efficiency and protein stability by immunoprecipitation and pulse-chase experiments indicated that a fourfold change in protein synthesis rate and a sevenfold rise in protein stability are responsible for the increase of TK expression. Progressive deletion of three, six, ten and 20 carboxy-terminal residues of the enzyme resulted in a stepwise loss of its growth-dependent regulation. In addition, a truncated protein lacking the last 30 amino acid residues was expressed at a level tenfold higher than the full-length polypeptide. Further analysis showed that removal of the C-terminal 30 residues did not affect the translation rate, but resulted in the drastic increase in protein half-life. These results demonstrate that residues at the carboxy terminus of the murine enzyme are essential for the growth-dependent regulation of TK protein stability.