Abstract
Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650-12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergent-resistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nm) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B' regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B'-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B' subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B' and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.
Highlights
The specificity of its immunoreactivity was confirmed on immunoblots of cyclin G1-glutathione S-transferase (GST), G2-GST, and GST alone and in parallel assays with anti-cyclin G2-NT antibodies raised against a peptide derived from NH2-terminal sequences of cyclin G2
Like full-length cyclin G2, expression of the cyclin G2GFP chimeras containing residues 142–241 resulted in a G1 phase cell cycle arrest. These findings were further confirmed by BrdUrd incorporation; cells expressing the NH2-terminal 1–140 residues of cyclin G2 had normal levels of BrdUrd incorporation, but cells expressing residues 142–344 of cyclin G2 exhibited low levels of DNA synthesis. These results suggest that the negative effects of ectopic cyclin G2 expression on cell cycle progression are linked to formation of a cyclin G2-phosphatase 2A (PP2A) complex
The preponderance of aberrant nuclei in cyclin G2 transfectants within 28 – 48 h post-transfection did not contain hypercondensed chromatin, were TUNEL-negative, and were surrounded by intact lamin B-positive nuclear envelopes. These observations suggest that the initial impact of unlicensed cyclin G2 expression was a dysregulation of a cellular division process leading to an aberrant mitosis/cytokinesis followed by cell cycle arrest rather than a direct induction of apoptosis
Summary
Protein phosphatase 2A (PP2A) is a highly conserved serine/threonine phosphatase essential for a plethora of cellular functions including signal transduction, translational control, endosome trafficking, and cell cycle regulation (16 –20). It is the chief target of various natural toxins and viral oncogenes [16, 21,22,23]. The diversity of PP2A function is paralleled by its requisite localization to distinct subcellular sites and specific targeting to a variety of cellular substrates, a crucial regulatory process for the proper function of many protein kinases [28]. The discovery that cyclin G1 interacts with PP2A BЈ subunits [44] implicates cyclin G1 in the regulation of PP2A and contrasts it with the classical cell cycle-promoting cyclins
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