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D-type cyclin-dependent kinase activity in mammalian cells
H Matsushime1, D E Quelle, S A Shurtleff
1Department of Genetics, University of Tokyo, Japan.
Abstract:
D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.
Insights
D-type cyclin-dependent kinase (CDK) activities, particularly those involving CDK4, were detected in mammalian cells. These kinases phosphorylate the retinoblastoma protein (pRb) and are regulated by cell cycle progression and serum stimulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- D-type cyclin-dependent kinase (CDK) activities were previously undetected in mammalian cells.
- Understanding the role of D-type cyclins and their partners in cell cycle regulation is crucial.
Purpose of the Study:
- To detect and characterize D-type cyclin-dependent kinase activities in mammalian cells.
- To investigate the regulation of these kinase activities during cell cycle progression.
Main Methods:
- Immune complex kinase assays using antibodies against cyclins D1, D2, D3, and CDK4.
- Analysis of kinase activity in rodent fibroblasts, mouse macrophages, and myeloid cells.
- Overexpression studies in engineered rodent fibroblasts.
Main Results:
- D-type cyclin-dependent kinase activities were detected, primarily associated with CDK4, readily phosphorylating retinoblastoma protein (pRb).
- Cyclin D1-CDK4 kinase activity emerged in mid-G1, peaked near the G1/S transition, and lagged behind cyclin induction.
- Activity remained dependent on serum stimulation even with enforced cyclin and CDK4 overproduction, suggesting regulation by upstream factors.
Conclusions:
- Mammalian cells possess D-type cyclin-dependent kinase activities, mainly mediated by CDK4-cyclin D complexes.
- The assembly of active cyclin D-CDK4 complexes and their kinase activity are tightly regulated by cell cycle entry and serum stimulation.
- Upstream regulators are essential for governing the formation of active D-type cyclin-CDK complexes.