Defining the substrate specificity of cdk4 kinase-cyclin D1 complex

cdk4 kinase-cyclin D1 complex (cdk4/D1) does not phosphorylate all of the sites within retinoblastoma protein (Rb) equally. Comparison of five phosphorylation sites within the 15 kDa C domain of Rb indicates that Ser795 is the preferred site of phosphorylation by cdk4/D1. A series of experiments has...

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Bibliographic Details
Published in:Carcinogenesis (New York) 1999-02, Vol.20 (2), p.193-198
Main Authors: GRAFSTROM, R. H, WEIJUN PAN, HOESS, R. H
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Analytical, structural and metabolic biochemistry
Arginine - metabolism
Binding Sites
Biological and medical sciences
Conserved Sequence
Cyclin D1 - metabolism
Cyclin-Dependent Kinase 4
Cyclin-Dependent Kinases - metabolism
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
HeLa Cells
Humans
Nuclear Proteins - metabolism
Phosphorylation
Proto-Oncogene Proteins
Retinoblastoma Protein - metabolism
Retinoblastoma-Like Protein p107
Serine - metabolism
Substrate Specificity
Threonine - metabolism
Transferases
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Summary:cdk4 kinase-cyclin D1 complex (cdk4/D1) does not phosphorylate all of the sites within retinoblastoma protein (Rb) equally. Comparison of five phosphorylation sites within the 15 kDa C domain of Rb indicates that Ser795 is the preferred site of phosphorylation by cdk4/D1. A series of experiments has been performed to determine the properties of this site that direct preferential phosphorylation. For cdk4/D1, the preferred amino acid at the third position C-terminal to the phosphorylated serine/threonine is arginine. Substitution of other amino acids, including a conservative change to lysine, has dramatic effects on the rates of phosphorylation. This information has been used to mutate less favorable sites in Rb, converting them to sites that are now preferentially phosphorylated by cdk4/D1. A conserved site at Ser842 in the related pocket protein p107 is also preferentially phosphorylated by cdk4/D1. Although Rb and p107 differ significantly in sequence, the Rb Ser795 site can replace the p107 Ser842 site without affecting the rate of phosphorylation. These results suggest that although a determinant of specificity resides in the sequences surrounding the phosphorylated site, the structural context of the site is also a critical parameter of specificity.
ISSN:0143-3334
1460-2180
DOI:10.1093/carcin/20.2.193