Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

BMC Cell Biology, Aug 2006

Background The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. Results We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of β-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Conclusion Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

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Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

BMC Cell Biology Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels Ke Yang 1 Yang Guo 1 William C Stacey 0 Jyoti Harwalkar 1 Jonathan Fretthold 1 Masahiro Hitomi 1 Dennis W Stacey 1 0 The Department of Neurology, Hospital of the University of Pennsylvania , Philadelphia, PA , USA 1 From the Department of Molecular Genetics, The Lerner Research Institute, The Cleveland Clinic Foundation , 9500 Euclid Avenue, Cleveland OH , USA Background: The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. Results: We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of -catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Conclusion: Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth. - Background Cyclin D1 plays a critical role in the regulation of proliferation by adjusting its expression levels to reflect the proliferative signaling environment of the cell, and then by regulating the cell cycle control machinery accordingly[1]. Cyclin D1 functions primarily to bind and activate the cyclin dependent kinase (CDK) 4/6, which then phosphorylates the retinoblastoma protein (Rb). Upon phosphorylation Rb releases the transcription factor E2F, which is then able to activate the transcription of genes required for G1/S phase transition[2-5]. The cyclin D1/CDK4/6 complex is also able to sequester p27kip1 and other CDK inhibitory proteins, thereby neutralizing their inhibitory capacity for cyclin E/CDK2[6] whose activity is required for G1/S transition[7,8]. The regulation of cyclin D1 activity is primarily dependent upon its expression level. This level is controlled by the regulation of gene expression, mRNA stability and translation, and by protein stability. Cyclin D1 mRNA synthesis is regulated by mitogenic signaling pathways downstream of Ras activity. These include the Raf-1, MEK1/2 and ERKs pathways[9-11] ; along with the Ral and Rac GTPases [12,13]. Translational control of cyclin D1 is also under the control of growth factor signaling through activation of the eukaryotic initiation factor 4E, an effector of the phosphatidylinositol-3 kinase (PI3K)/ AKT/mTOR signal pathway [14]. The stability of cyclin D1 protein also plays a major role in the regulation of its expression. Phosphorylation on Thr-286 has been reported to result in rapid proteasomal degradation of cyclin D1 [15]. It is also possible that this phosphorylation results in the export of cyclin D1 from the nucleus where it is functionally inactivated due to separation from its nuclear substrates [16]. In either case, the kinase responsible has been reported to be glycogen synthase kinase 3 (GSK3), which is an excellent in vitro kinase for cyclin D1 Thr-286 [17]. GSK3 is presumed to be constitutively active and therefore able to suppress cyclin D1 levels until phosphorylated. This phosphorylation can be carried out by AKT, which is in turn activated by PI3K [18,19], suggesting that the PI3K/AKT/GSK3 pathway controls cyclin D1 stability [15,17]. Not only are overall cyclin D1 levels critical in the growth properties of the cell, the levels of this protein are actively regulated through the cell cycle. We observed this fact using quantitative image analysis of antibody stained asynchronous cultures. Cyclin D1 expression was found to (...truncated)


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Ke Yang, Yang Guo, William C Stacey, Jyoti Harwalkar, Jonathan Fretthold, Masahiro Hitomi, Dennis W Stacey. Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels, BMC Cell Biology, 2006, pp. 33, 7, DOI: 10.1186/1471-2121-7-33