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G1 cyclins control the retinoblastoma gene product growth regulation activity via upstream mechanisms
L E Horton1, Y Qian, D J Templeton
1Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Abstract:
Inactivation of the retinoblastoma gene product (pRb) occurs concomitant with the appearance of its hyperphosphorylated form in mid to late G1. Multiple cyclin/CDK complexes are implicated in the cell cycle phosphorylation of pRb. Using in vivo expression systems, we show that cyclins A, E, D1, D2, and D3 each function to phosphorylate and inactivate pRb. In vivo, G1 cyclin/kinase complexes enhance the phosphorylation of pRb, and these effects of cyclin/kinases on pRb can be overcome by the addition of p21, a wide spectrum inhibitor of G1 kinases. Kinases associated with cyclins A, E, and D1 phosporylate pRb indistinguishably in vivo, according to proteolytic maps. Although cyclin D1 has been reported to bind to pRb directly, requiring the pRb-binding motif LXCXE, a mutant D1 lacking the pRb-binding motif remains able to phosphorylate pRb in vivo and in vitro and is also able to reverse the growth-inhibitory properties of pRb in intact cells. Finally, coexpression of G1 cyclins and kinases represses pRb-mediated growth inhibition in Saos-2 cells. The multiplicity of mechanisms for pRb phosphorylation and inactivation suggests that several pathways exist for the regulation of pRb by phosphorylation.
Insights
Multiple G1 cyclins (A, E, D1, D2, D3) phosphorylate and inactivate the retinoblastoma gene product (pRb). This process, crucial for cell cycle progression, can be modulated by p21, a G1 kinase inhibitor.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The retinoblastoma gene product (pRb) is a key regulator of cell cycle progression.
- pRb inactivation, through hyperphosphorylation, is essential for transitioning from the G1 to S phase.
- Cyclin-dependent kinases (CDKs) complexed with cyclins are known regulators of pRb phosphorylation.
Purpose of the Study:
- To investigate the specific roles of various G1 cyclins in pRb phosphorylation and inactivation.
- To determine if cyclin D1's interaction motif is essential for pRb phosphorylation.
- To explore the potential for modulating pRb activity through cyclin/kinase complexes.
Main Methods:
- In vivo expression systems were utilized to study cyclin function.
- Proteolytic mapping was employed to analyze pRb phosphorylation patterns.
- In vitro kinase assays and cell-based assays (Saos-2 cells) were performed.
Main Results:
- Cyclins A, E, D1, D2, and D3 were shown to phosphorylate and inactivate pRb in vivo.
- G1 cyclin/kinase complexes enhance pRb phosphorylation, an effect counteracted by p21.
- Mutant cyclin D1 lacking the pRb-binding motif retained phosphorylation and growth inhibition reversal capabilities.
- Kinases associated with cyclins A, E, and D1 phosphorylated pRb similarly in vivo.
Conclusions:
- Multiple G1 cyclins contribute to pRb phosphorylation and inactivation, indicating diverse regulatory pathways.
- The pRb-binding motif on cyclin D1 is not essential for its ability to phosphorylate pRb.
- Modulating G1 cyclin/kinase activity offers a potential mechanism for controlling pRb-mediated growth inhibition.