Product Pathways - Cell Cycle / Checkpoint
Rb-C Fusion Protein #6022
Molecular Weight
76 kDa
Description
Rb-C (retinoblastoma protein C-terminus) Fusion Protein serves as a useful substrate for various cyclin-dependent kinases (CDK's) (8,9,11). It is expressed as a recombinant protein fusion of Rb residues 701?928 and maltose binding protein. The phosphorylation sites present in this C-terminal portion of Rb include Ser780 (8), Ser795 (9) and Ser807/811 (11).
Directions for Use
Rb-C Fusion Protein at a concentration of 2 µg/20 µl reaction can be phosphorylated using cdc2 Protein Kinase (NEB #P6020) in an in vitro kinase assay with 1X Kinase Buffer #9802 and 200 µM ATP #9804. After 30 minutes at 30°C, phosphorylation can be detected by Western blot with Phospho-Rb (Ser795) Antibody #9301.
Background
The retinoblastoma tumor suppressor protein, Rb, regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).
- Sherr, C.J. (1996) Science 274, 1672-1677.
- Nevins, J.R. et al. (1992) Science 258, 424-429.
- Welch, P.J. and Wang, J.Y. (1993) Cell 75, 779-790.
- Hu, Q.J. et al. (1990) EMBO J. 9, 1147-1155.
- Knudsen, E.S. and Wang, J.Y. (1997) Mol. Cell. Biol. 17, 5771-5783.
- Lundberg, A.S. and Weinberg, R.A. (1998) Mol. Cell. Biol. 18, 753-761.
- Connell-Crowley, L. et al. (1997) Mol. Cell. Biol. 8, 287-301.
- Kitagawa, M. et al. (1996) EMBO J. 15, 7060-7069.
- Geng, Y. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 194-199.
Application References
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