Functional analysis at the Cys706 residue of the retinoblastoma protein.

R.A Kratzke,G.A Otterson,A.Y Lin,E Shimizu,N Alexandrova,M Zajac-Kaye,J.M Horowitz,F.J Kaye

doi:10.1016/s0021-9258(18)35707-7

R.A Kratzke, G.A Otterson + Show 6 more

Open Access

https://doi.org/10.1016/s0021-9258(18)35707-7

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Abstract

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.

Highlights

From the $Navy Oncology Branch and §Medicine Branch,National Cancer Institute, Bethesda, Maryland 20889 and the Wection of Cell Growth, Regulation. and Oncogenesis and the Departmentof Microbiology and Immunology, Duke University Medical Center, Durham, NorthCarolina 27710
The Cys706Residue Does Not Possess Intrinsic Activity for (Perkin Elmer-Cetus, Norwalk, CT) except that 10% dimethyl sulf- Oncoprotein Binding-To examine whether the Cys706residue oxide was added to the reaction mixtures
Oncoprotein Binding to ElA and T Antigen Using in Vitro Transit was previously hypothesized that Cys706might participate in a disulfide bond [21,22], we individually mutated two upstream cysteine residues that resided within the RB pocket lated RB-PlasmidDNAwas linearized and subjected to in vitro at codons 407 and 489

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

Vol 267,No.36,Issue ofDecember 25,pp. 25998-26003,1992 Printed in U.S.A. Robert A. This splicing mutant has been identified in tumors from differing histologic types including bladder, lung, andprostate cancers [18,19,20] Another RB mutant protein,defective in phosphorylation and oncoprotein binding, was identified with a point mutationwithin exon 21 resulting in a single cysteine to phenylalanine substitution at codon 706 [21, 22]. + Phe RB mutant protein from cell line H209 with compa- vitro phosphorylation assays, using immunoprecipitated p34"* This finding suggests impor- ~34'~'', Oncogene Science) from "phase arrested HeLa cells, were t a n t differences between MYC and the other RB-associated performed as described previously [35], except that equal amounts of proteins in their binding patterns to theRB product. Oligonucleotides spanning aninternal DraIIIrestriction nuclease site at nucleotide position 2129.All nucleotide coordinates for the RB

RESULTS

Analysis of Mutant RR Protein

DISCUSSION