DNA-binding activity of retinoblastoma protein is intrinsic to its carboxyl-terminal region.
The retinoblastoma (RB) gene encodes a nuclear phosphoprotein with a molecular weight of 110,000 (pp110RB) associated with DNA-binding activity. This sequence-nonspecific DNA binding activity was further studied by Southwestern and DNA-cellulose chromatography using purified fusion proteins expressed in Escherichia coli. Three fusion proteins, containing amino acids 612-775, 776-928, and 612-928 of pp110RB, bound to DNA; the binding affinity of the latter was approximately 20-fold higher than those of either smaller region. Other regions of pp110RB had no detectable binding activity, indicating that the carboxyl-terminal region of the RB protein is the major domain responsible for interacting with DNA. Since several potential phosphorylation sites reside within this region, isoforms of RB protein from cellular lysates with various degrees of phosphorylation were compared with respect to their DNA-binding affinity. The hyperphosphorylated form was eluted from DNA-cellulose columns at 0.1-0.3 M NaCl, whereas the hypophosphorylated form appeared in the eluates only at salt concentrations of 0.4-0.7 M, implying that phosphorylation of RB protein may affect its DNA-binding activity. That pp110RB can bind DNA intrinsically, and that this activity can be modulated by phosphorylation, is consistent with the proposed regulatory role of the RB protein in cell growth and differentiation.
Wang
N P
NP
Department of Pathology, Center for Molecular Genetics, University of California, San Diego, La Jolla 92093.
Chen
P L
PL
Huang
S
S
Donoso
L A
LA
Lee
W H
WH
Lee
E Y
EY
D015139:Blotting, Southern; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics*
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D004247:DNA