Functional analysis at the Cys706 residue of the retinoblastoma protein.
A missense mutation at cysteine 706, resulting in a retinoblastoma (RB) protein defective in phosphorylation and oncoprotein binding, has been isolated from a human tumor cell line. Since this residue is conserved in murine RB and in the related p107 protein, we studied the activity of in vitro mutants flanking this position. These experiments demonstrated that the thiol atom at codon 706 does not possess intrinsic functional activity as small polar or nonpolar residues could substitute at either codons 706 or 707, while bulkier R-group changes in these positions interfered with in vitro oncoprotein binding or in vivo protein phosphorylation. A series of missense mutants in an adjacent leucine repeat domain also demonstrated a loss of oncoprotein binding that was proportional to the magnitude of amino acid substitutions. To determine whether the cysteine 706 --> phenylalanine RB mutant retained any protein binding activity, we examined its ability to precipitate MYC, which was recently identified as a potential RB-associated protein. These experiments demonstrated that the mutant RB product is capable of binding in vitro to c-myc and L-myc proteins with comparable affinity as wild-type RB. These findings raise questions about the functional role of the RB:MYC interactions and emphasize important differences in the binding patterns between MYC and the other RB-associated proteins.
Kratzke
R A
RA
Navy Oncology Branch, National Cancer Institute, Bethesda, Maryland 20889.
Otterson
G A
GA
Lin
A Y
AY
Shimizu
E
E
Alexandrova
N
N
Zajac-Kaye
M
M
Horowitz
J M
JM
Kaye
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D018288:Carcinoma, Small Cell / Q000235:genetics*; D003062:Codon; D003545:Cysteine; D005091:Exons; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D003062:Codon; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D005982:Glutathione Transferase; D003545:Cysteine