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 detergentresistant 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.

The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.

Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition, and the formation of aberrant nuclei [1]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT), and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells, and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome, resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs, and a p53 dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFPcentrin tagged centrioles, the mature centriole present at microtubule foci, indicate that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology, and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities.

The mammalian target of rapamycin (mTOR) is a central component within a complex intracellular signaling network that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. A hyperactive PI3k/Akt/mTOR signaling pathway is found in many human cancers and alterations in this pathway is associated with the development and progression of cancer. Drugs that target and inhibit mTOR activity are therefore expected to provide therapeutic value in a number of cancer types. Several classes of mTOR-targeted therapeutics are currently being evaluated in cancer clinical trials, including the rapamycins, dual PI3K-mTOR inhibitors, and ATP-competitive mTORC1/2 inhibitors. This review summarizes important findings from recently completed trials of mTOR inhibitors and also discusses preliminary data from ongoing trials.

The adult central nervous system (CNS) has a remarkable ability to repair itself. However, severe brain and spinal cord injuries cause lasting disability and there are only a few therapies that can prevent or restore function in such cases. In this review, we provide an overview of traumatic CNS injuries and discuss several emerging pharmacological options that have shown promise in preclinical and early clinical studies. We highlight therapies that modulate mammalian target of rapamycin (mTOR) signaling, a pathway that is well known for its roles in cell growth, metabolism and cancer. Interestingly, this pathway is also gaining newfound attention for its role in CNS repair and regeneration.

Ligand activated estrogen receptors (ER) drive growth of nearly two-thirds of breast cancers (BC). Acquired BC tumor resistance to endocrine-based therapeutics that inhibit ER signaling poses a significant challenge for long-term abatement of ER-positive BCs. Signaling crosstalk between activated ER and peptide growth factor receptor pathways (e.g. HER2 and IGF-1R) is one adaptive mechanism promoting ER-positive BC tumor resistance to drugs inhibiting estrogen (E2) signaling. Definition of the cell cycle control proteins that modify tumor cell resistance to the ER-antagonists tamoxifen and fulvestrant could improve future therapeutic approaches for control of BC. Cyclin G2 (CycG2) is an unconventional cyclin upregulated during cell cycle arrest responses to a variety of cellular stresses and growth inhibitory conditions. Transcription of the gene encoding CycG2, CCNG2, is directly repressed by E2-bound ER complexes in BC cell lines. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2 overexpressing BC cells, and that ectopic CycG2 expression induces cell cycle arrest in BC cell lines. Here we show that pharmacological blockade of ER-signaling in the E2-dependent BC cell lines MCF-7 and T-47D enhances the expression and nuclear localization of CycG2. shRNA-mediated knockdown of CycG2 in E2-deprived and fulvestrant-treated MCF-7 cells dampened the cell cycle arrest response to these treatments. Our evidence suggests that loss of CycG2 increases phospho-activation of MEK1 and inhibitory phosphorylation of RB. Interestingly our work also indicates that CycG2 can form complexes with CDK10, an orphan CDK linked to inhibition of tamoxifen resistance and modulation of RAF/MEK/MAPK signaling. Recently we reported that signaling through insulin and insulin-like growth factor-1 receptors (IGF-1R) strongly represses CycG2 expression. Current studies suggest that the anti-diabetic drug metformin (MTFN) inhibits BC cell growth by promoting AMPK-mediated suppression of growth factor receptor activation of mTOR. Patients taking MTFN exhibit a dose-dependent reduction in cancer risk and clinical trials indicate that BC therapies including MTFN improve response rates. We found that MTFN treatment stimulates CycG2 expression and potentiates both fulvestrant-mediated upregulation of CycG2 expression and growth-inhibition of MCF-7 cells. Moreover knockdown of CycG2 expression blunts the enhanced anti-proliferative effect of MTFN on fulvestrant treated cells. Importantly, analysis of BC tumor cDNA microarray databases indicates that CCNG2 transcripts are reduced in aggressive, poor-prognosis BCs and that higher levels of CCNG2 expression in the patient samples correlated with longer periods of relapse free survival. Citation Format: Mary C. Horne, Maike Zimmerman, Aruni S. Arachchige Don, Michaela Donaldson, Tommaso Patriarchi. Knockdown of cyclin G2 expression hinders the cell cycle arrest response of MCF-7 cells to estrogen receptor signaling-antagonists and treatment with the antidiabetic metformin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2014-4559

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