Carboxy-terminal residues of mouse thymidine kinase are essential for rapid 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 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.
Sutterluety
H
H
Institute of Molecular Biology, University of Vienna, Austria.
Bartl
S
S
Karlseder
J
J
Wintersberger
E
E
Seiser
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D000939:Epitopes; D005347:Fibroblasts; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis; D017384:Sequence Deletion; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D013937:Thymidine Kinase