Identification and characterization of a novel human cDNA encoding a 21 kDa pRb-associated protein.
The retinoblastoma protein (pRb) functions as a critical master regulator in cell cycle regulation, which is an important cell-regulatory process, through its interaction with various cellular proteins. Using the C-terminus of human pRb and the yeast two-hybrid system, a novel protein named RBP21 that contains 187 amino acid residues with a calculated molecular weight of 21 kDa was identified as a pRb-binding protein. Sequence analysis indicates that RBP21 shares homology with other retinoblastoma-binding proteins in the pRb-binding motif LxCxE at the C-terminal region. In vitro specific interaction between pRb and RBP21 was confirmed using in vitro translation products. When overexpressed in COS-7 cells, RBP21 could co-immunoprecipitate with pRb. This interaction requires the LxCxE motif of RBP21 and the entire pocket region of pRb. Each point mutation of the conserved amino acid residues in pRb-binding motif of RBP21 abolished its specific interaction with pRb. RH mapping result showed that this novel gene was mapped to chromosome region 15q21.1-21.3. Northern blot analysis suggested that RBP21 was widely expressed in various human tissues and cancer cell lines. When expressed in HeLa cells as a green fluorescent protein fusion, RBP21 was distributed throughout the cell.
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State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China.
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D017500:Adenovirus E1A Proteins; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins; D002874:Chromosome Mapping; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D017500:Adenovirus E1A Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C416925:EID1 protein, human; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D049452:Green Fluorescent Proteins