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A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein
F J Kaye1, R A Kratzke, J L Gerster
1National Cancer Institute-Navy Medical Oncology Branch, Naval Hospital, Bethesda, MD 20814.
Abstract:
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.
Insights
Researchers found a specific mutation in the retinoblastoma protein (RB1) in small-cell lung cancer cells. This RB1 mutation prevents normal protein function and interaction with viral proteins.
Area of Science:
- Oncology
- Molecular Biology
- Protein Biochemistry
Background:
- Small-cell lung cancer (SCLC) cell lines can exhibit aberrant retinoblastoma protein (RB1) expression.
- The NCI-H209 SCLC cell line displays an underphosphorylated RB1 form.
Purpose of the Study:
- To investigate the molecular basis of the aberrant RB1 form in NCI-H209 cells.
- To determine the functional consequences of a specific RB1 mutation.
Main Methods:
- Molecular analysis of RB1 mRNA to identify mutations.
- Site-directed mutagenesis to create mutant RB1 cDNA.
- Stable expression of mutant RB1 in RB1-deficient human cells.
- Assessment of RB1 phosphorylation status.
- Analysis of RB1 protein-protein interactions with viral oncoproteins.
Main Results:
- A single point mutation (cysteine to phenylalanine) was identified in exon 21 of RB1 in NCI-H209 cells.
- This cysteine-to-phenylalanine substitution at codon 706 inhibited RB1 phosphorylation.
- The mutation abolished RB1 binding to SV40 large tumor antigen and adenovirus E1A.
Conclusions:
- The identified RB1 mutation in exon 21 is sufficient to cause functional defects.
- Cysteine at codon 706 is critical for maintaining RB1 conformation and protein-protein interactions.
- This finding highlights the importance of specific RB1 residues in tumor suppressor function.