Incorrect maintenance of genetic material leads to a majority of human diseases. Genetic errors can range from single nucleotide mutations in essential protein coding genes to the rearrangement or loss/gain of entire chromosomes. Cells are most vulnerable to these sorts of errors during the cell division cycle when the entire genome is duplicated and segregated to two daughter cells. The progress from each phase of the cell cycle is regulated by a host of proteins with cyclin-CDK complexes serving as the main drivers. Cyclin-dependent kinases (CDKs) drive the cell cycle transitions by phosphorylating many targets; however, as the name implies, they can only do this when bound to the appropriate cyclin. The expression and degradation of cyclin genes/proteins are tightly controlled to protect genetic integrity. For example, if cyclin D is produced too early, cells may enter the cell cycle despite nutrient deficits, or if cyclin B is degraded too soon cells exit the cell cycle without equally segregating the chromosomes to each daughter cell. At Fred Hutch, many researchers are working to understand how different cancers manipulate the cell cycle, often to increase their growth rate. The laboratory of Dr. Bruce Clurman (Human Biology and Clinical Research Divisions) is interested in transitions into and out of the DNA replication stage of the cell cycle, S-phase. Thru these studies Ryan Davis, a graduate student in the Clurman Lab, recently found that the phosphatase PP2A-B56 plays a role in extending the lifetime of the S-phase associated cyclin E. This study, published in Molecular and Cellular Biology, uncovered new aspects of cyclin E regulation by this phosphatase and implicated it as a possible oncogene.
Cyclin E is phosphorylated at three amino acids, Thr62, Thr380, and Ser384, this event stimulates the degradation of cyclin E. The kinases responsible are fairly well known; however the phosphatases involved and the rates of phosphorylation/deposphorylation at these sites remained largely unknown. This is largely due to technical difficulties - cyclin E degradation after phosphorylation of Ser384 is immediate, making this modification undetectable by standard immunoblot. In this study Davis overcame this challenge by decreasing FBXW7 activity, which ubiquitinates cyclin E and targets it for degradation. This was achieved in three different cell types using both RNAi depletion and deletion of the FBXW7 gene. The FBXW7 mutations stabilized cyclin E protein sufficiently so that it could be detected by immunoblot. With these tools in hand, researchers measured phosphorylation of Thr62, Thr380, and Ser384 before and after exposure to kinase or phosphatase inhibitors. First, cells were treated with Calyculin A, a broad-spectrum phosphatase inhibitor that targets many phosphatase enzymes. Calyculin A treatment for 5-20 minutes did not change phosphorylation level of Thr62 or Thr380, but S384 phosphorylation levels increased. This suggests that S384 is dephosphorylated by either PP1 or PP2. Second, cells were treated with Roscovitine, an inhibitor that specifically targets CDK2, the kinase for Ser384. Similar to Calyculin A, 5-20 minute treatments with Roscovitine caused a decrease in phosphorylation of cyclin E Ser384. This indicated that Ser384 is rapidly phosphorylated by CDK2 and targeted for degradation, but one of the PP1 or PP2 phosphatases can counteract this to stabilize cyclin E levels.
Many currently available phosphatase inhibitors target a spectrum of human phosphatases so Davis and colleagues used siRNA to target specific phosphatases and pinpoint which enzyme dephosphorylated cyclin E Ser384. An initial screen targeted PP1α, PP1β, PP1γ, PP2A, PP4, PP5, or PP6. These proteins were depleted by siRNA then cell lysates were mixed with purified cyclin E to measure in vitro dephosphorylation of cyclin E. Cyclin E Ser384 phosphorylation was elevated only when PP2A was depleted, indicating this is the enzyme that counteracts kinase activity. This first pass targeted the catalytic subunit of PP2A, however this subunit pairs with one of at least a dozen ‘B’ subunits to form a functional holoenzyme. Using the same approach Davis revealed that lysates depleted of PP2A-B55α, -B56γ, and -B56δ did not dephosphorylate cyclin E in vitro. A large function of the ‘B’ subunits is to direct the sub-cellular localization of PP2A, a function that is lost in vitro, and thus researchers validated these findings in cells. In this case ‘B’ subunits were highly overexpressed and interestingly only PP2A-B56 (α, β, γ, ε) and not PP2A-B55 subunits caused a decrease Ser384 phosphorylation.
As a cell cycle protein, cyclin E is synthesized in the cell (and thus regulated) at the end of G1 thru S phase. While cyclin E should not be present in other cell cycle phases that can occur in cells lacking FBXW7, so Davis and colleagues asked if PP2A regulation of cyclin E could occur at any cell cycle stage. When cells were treated with a microtubule poison to arrest them in mitosis Roscovitine treatment did not alter Ser384 phosphorylation, indicating PP2A-B56 could not dephosphorylate it.
This role for PP2A was likely not appreciated previously because cyclin E degradation is so rapid. Thus it will be important to understand these kinetics in FBXW7 positive cells, yet Davis explained this finding may have uncovered interesting tumor biology, "One of the things that we are most excited about exploring in future work is the potential link between PP2A-B56 amplifications and cyclin E activity in human cancers." Both Cyclin E and PP2A-B56 are genetically amplified in breast and ovarian cancers. This likely contributes to the hyper-proliferative nature of these tumor types by reducing the duration of the cell cycle and may represent an exciting therapeutic target. The relevance of these two enzymes would have been missed without these findings.
Davis RJ, Swanger J, Hughes BT, Clurman BE. (2017). The PP2A-B56 phosphatase opposes cyclin E autocatalytic degradation via site-specific dephosphorylation. Mol Cell Biol.
Funding for this research was provided by the National Cancer Institute (NIH) and National Science Foundation.