Abstract
Cell cycle progression through its regulatory control by changes in intracellular Ca2+ levels at the G1/S transition mediates cellular proliferation and viability. Ca2+/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr157 and Thr198 in human cells and at Thr170 and Thr197 in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy.
Highlights
During cell replication, the G1 phase is critical and regulated by integration of a variety of signals that determine whether the cell is destined to proliferate, differentiate, or die [1]
We demonstrate that i) CaM-dependent kinase 1 (CaMKI) phosphorylates p27 at Thr157 and Thr198 in human cells and at Thr170 and Thr197 in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression
CaMKI protein was most efficiently degraded by Fbxl12, a protein upregulated during TGF-β-induced G1 arrest [4]
Summary
The G1 phase is critical and regulated by integration of a variety of signals that determine whether the cell is destined to proliferate, differentiate, or die [1]. The SCF complexes have emerged as important modulators of cell cycle progression in normal, transformed, or malignant cells via degradation of key regulatory proteins such as G1-phase cyclins, cyclin dependent kinase (cdk) inhibitors, transcription factors and others. In this SCF complex, the F box protein confers substrate recognition specificity. The F-box protein, Fbxo, was shown to initiate G1 arrest via cyclin D1 degradation after DNA damage caused by γ-irradiation [3] Another related protein, Fbxl, mediates osteoblast cell differentiation by mediating p57kip ubiquitin-proteasome degradation [4]. Unlike other SCF F- box proteins that usually target phosphodegrons, the recently described Fbxl protein targets cyclin D2 [5] or cyclin D3 [6] via recognition of a canonical calmodulin (CaM)-binding motif that induces Go or G2/M arrest respectively
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