CARBOXY-TERMINAL RESIDUES OF MOUSE THYMIDINE KINASE ARE ESSENTIAL FORRAPID DEGRADATION IN QUIESCENT CELLS

The expression of murine thymidine kinase (TK) is highly dependent on the growth state of the cell. The enzyme is nearly undetectable in res ting (G(0)) 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 wit h the coding region of the TK cDNA under the control of a constitutive SV40 promoter. While TK mRNA levels were growth independent in this c ell line, TK protein expression and enzyme activity were low in restin g cells but increased strongly after growth stimulation by serum. Meas urements of translation efficiency and protein stability by immunoprec ipitation and pulse-chase experiments indicated that a fourfold change in protein synthesis rate and a sevenfold rise in protein stability a re responsible for the increase of TK expression. Progressive deletion of three, six, ten and 20 carboxy-terminal residues of the enzyme res ulted in a stepwise loss of its growth-dependent regulation. In additi on, a truncated protein lacking the last 30 amino acid residues was ex pressed at a level tenfold higher than the full-length polypeptide. Fu rther analysis showed that removal of the C-terminal 30 residues did n ot affect the translation rate, but resulted in the drastic increase i n protein half-life. These results demonstrate that residues at the ca rboxy terminus of the murine enzyme are essential for the growth-depen dent regulation of TK protein stability. (C) 1996 Academic Press Limit ed