G{sub 1} arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637
- Univ. of California, Davis, CA (United States)
- Univ. of Texas, Houston, TX (United States); and others
The protein kinase inhibitor staurosporine has been shown to induce G{sub 1} phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G{sub 1} phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB{sup {minus}}). However, when infected with a pRB-expressing retrovirus, these cells, now pRB{sup +} and pRB{sup {minus}} cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cylin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB{sup +} cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21{sup (Wafl/Cipl)} and p27{sup (Kipl}) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p21 form staurosporine-treated pRB{sup +} cells revealed a 2.5- to 3-fold higher ratio of p21 bound to CDK2 compared with staurosporine-treated pRB cells. In pRB{sup +} cells, p21 was preferentially associated with Thr160 phosphorylated active CDK2. In pRB{sup {minus}} cells, however, p21 was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G{sub 1} arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB-dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p21 and p27. 47 refs.
- DOE Contract Number:
- W-7405-ENG-36
- OSTI ID:
- 457015
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 12 Vol. 93; ISSN 0027-8424; ISSN PNASA6
- Country of Publication:
- United States
- Language:
- English
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