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Cyclin-dependent kinase-2 (Cdk2) forms an inactive complex with cyclin D1 since Cdk2 associated with cyclin D1 is not
H Higashi1, I Suzuki-Takahashi, S Saitoh
1Banyu Tsukuba Research Institute, Japan.
Abstract:
Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
Insights
Cyclin-dependent kinase 2 (Cdk2) forms inactive complexes with cyclin D1. This complex lacks kinase activity because Cdk2 is not phosphorylated by Cdk7-cyclin-H, preventing its activation and binding to cyclin D1.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin-dependent kinases (Cdks) complex with cyclins to regulate cell cycle progression.
- Cdk2, complexed with cyclins A and E, phosphorylates targets like pRB, driving cell cycle progression.
- The kinase activity of Cdk2-cyclin D1 complexes remains unclear, with suggestions of inactivity.
Purpose of the Study:
- To investigate the kinase activity of Cdk2-cyclin D1 complexes.
- To elucidate the reasons behind the potential lack of activation in Cdk2-cyclin D1 complexes.
- To determine the role of Cdk2 phosphorylation in its interaction with cyclins.
Main Methods:
- Produced Cdk, cyclin, and Cdk-cyclin complexes using a baculovirus expression system.
- Purified Cdk2-cyclin D1 complexes and assessed their kinase activity against various substrates (H1 histone, pRB, etc.).
- Analyzed Cdk2 phosphorylation status and its interaction with cyclins in both baculovirus systems and human WI-38 cells.
Main Results:
- The purified Cdk2-cyclin D1 complex exhibited no kinase activity against tested substrates, unlike Cdk2-cyclin E and Cdk2-cyclin A complexes.
- Inactive, under-shifted Cdk2 was detected in the Cdk2-cyclin D1 complex, indicating a lack of activation.
- Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, and phosphorylated Cdk2 did not bind to cyclin D1.
Conclusions:
- Cdk2 forms a stable but inactive complex with cyclin D1.
- The lack of Cdk2 activation in the Cdk2-cyclin D1 complex is due to the absence of Cdk7-cyclin-H-mediated phosphorylation.
- Phosphorylation of Cdk2 by Cdk7-cyclin-H is essential for its activation and binding to cyclins like cyclin E, but not cyclin D1.