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.
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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)