McArdle Blog L7 ~ 10/01/2013
The McArdle Papers: Discovering the structure of the tumor suppressor PP2A and its implications in regulating DNA synthesisThe cells that make up our bodies normally grow and divide in an extremely well regulated and coordinated fashion. There are many pathways that control this process, and disruptions at key points may allow for unchecked growth of cells. Cancers can result when normal cells start growing uncontrollably. There are many different causes and types of cancer because these pathways that regulate cell growth and division are incredibly complex and there are many points where they can fail.
One of the more common mechanisms by which a cell regulates normal cell division is by reversible phosphorylation of proteins. Phosphorylation is a type of modification where a phosphate (PO4) group is added to specific amino acids at precise places in a protein. This modification is typically carried out by a class of proteins called kinases and reversed by proteins called phosphatases. Very generally speaking, proteins phosphorylated by kinases tend to activate pathways for growth and division. On the other hand, dephosphorylation (i.e. removal of the phosphate group by phosphatases) of these proteins tends to stop or prevent these pathways from being activated (see the image below). It should be no surprise then that kinases tend to be overproduced or have higher activity in cancer cells, and phosphatases tend to be inhibited. Kinases have been well studied, and some chemotherapy drugs that target kinases have been used successfully, but none of them have proven to be the “silver bullet” against cancer that many hoped they would be.
Phosphatases have been studied far less, mostly due to their complex regulation and activity. While there are over 400 kinases, there are far fewer phosphatases, and our understanding of the mechanisms by which they interact with their substrates (i.e. target proteins) are mostly based on the three dimensional structure of the phosphatase or the phosphatase-substrate complex. A better understanding of the mechanisms by which phosphatases interact with their substrates is critical if either of these molecules is to be targeted for cancer treatments.
One of the proteins critical in the proper control of cell growth and division is Cell Division Control 6 (CDC6). CDC6 forms a complex with other proteins on DNA and signals to the DNA synthesis machinery to begin DNA replication at certain origins. The proper placement and timing of this complex is necessary for accurate and controlled DNA replication during cell division. Phosphorylation by kinases target and activate this protein, and dephosphorylation leads to its degradation. The phosphatase that does this is Protein Phosphatase 2A (PP2A). This phosphatase consists of three variable parts, different combinations of which confer useful enzymatic activity on specific molecules with phosphate modifications. The three parts, which together form an active holoenzyme, each have different functions. The catalytic subunit, or C subunit, contains the active site and performs the actual cleavage of the phosphate, the regulatory subunit, or B subunit, confers substrate specificity to the holoenzyme, and the scaffold, or A subunit, unites these two together. Various combinations of subunits allow PP2A to target a wide array of phosphorylated substrates. Being able to visualize the three dimensional structure of this protein is an important step in determining its mechanism of action and possible drug targeting sites. It is also useful to know if there are ways to alter the activity and binding of the protein both with itself and with its substrates.
There are approximately 30 different PP2A holoenzymes, but we know the structures for only two of them, and for none in the same family of regulatory subunits as PR70. We determined the PP2A-PR70 holoenzyme structure at high atomic resolution using x-ray crystallography (Figure 1). We were able to visualize the interactions between the three subunits and use mutational analyses to confirm that disruptions in these regions could cause the holoenzyme to fail to assemble or to not have activity against CDC6. These key mutations that disrupt the structural and functional integrity of P2A may be useful markers to search for in cancers where CDC6 activity is not properly regulated.
When we compared the structure of PP2A-PR70 that we determined to structures determined by others, and performed biochemical measurements, we found that the scaffold subunit (A) has a large amount of flexibility, and the PR70 subunit causes the holoenzyme to be more compact than any of the other PP2A holoenzymes seen to date (Figure 2).
This flexibility of the A subunit is also important for allowing PR70 to interact directly with the catalytic subunit, which is necessary for CDC6 dephosphorylation. We also provide the first direct evidence that PR70 is the subunit that is specifically responsible for CDC6 dephosphorylation and that other regulatory subunits have no activity against it (Figure 3). This is important because disruptions in the assembly of PP2A holoenzymes with the proper regulatory subunits – such as R70 – at the correct time in the cell cycle may result in uncontrolled cell division and may be a mechanism that leads to transformation or maintenance of cancerous cells.
We hope that this research has added important knowledge of the structure and function of a little studied, but important, member of the PP2A family. Knowing this information may lead to identifications of at-risk mutations, targets for environmental chemicals or pharmaceuticals which may help maintain proper function or repair aberrant function of the PP2A holoenzyme. This discovery also expands our knowledge on the workings of the cell cycle and provides potential avenues for therapeutic developments in cancer prevention and treatment.
These findings were first reported in Wlodarchak N., et. al. 2013. Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation. Cell Research 23(7) p931-946