A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding.
We have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.
Kaye
F J
FJ
National Cancer Institute-Navy Medical Oncology Branch, Naval Hospital, Bethesda, MD 20814.
Kratzke
R A
RA
Gerster
J L
JL
Horowitz
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D018288:Carcinoma, Small Cell; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics* / Q000302:isolation & purification; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D016160:Retinoblastoma Protein; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016160:Retinoblastoma Protein