Since its discovery, phosphorylation has come to be recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in a host of human diseases.
It is no coincidence that one cellular process is mentioned time and time again in discussions of cell-signaling pathways in cancer. Since its discovery, phosphorylation has come to be recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in a host of human diseases.
In this report, we probe a little deeper to understand what exactly protein phosphorylation does; why it is such a vital, ubiquitous process; and how it continues to further our understanding of diseases such as cancer.
Once a gene is expressed and translated into a functional cellular protein, the cell is able to control the protein’s fate through the use of posttranslational modifications (PTMs). Phosphorylation is the most important and most thoroughly researched form of PTM.
Phosphorylation of a protein involves the enzymatically mediated addition of a phosphate group (PO4) to its amino acid side chains. Phosphorylated proteins were observed as far back as the early 1900s, but it was not until the 1950s that the pioneering, and ultimately Nobel Prizewinning, discoveries of Edmond H. Fischer and Edwin G. Krebs determined that phosphorylation was a reversible, enzymatically mediated process, capable of modifying the function of a protein.
Today, it is believed that as many as one-third of all proteins in the cell are phosphorylated at one time or another, and half of these proteins likely harbor more than 1 phosphorylation site, with different sites often eliciting quite different cellular responses.
Phosphorylation and the reverse reaction, dephosphorylation, occur thanks to the actions of 2 key enzymes. Protein kinases phosphorylate proteins by transferring a phosphate group from adenosine triphosphate (ATP) to their target protein. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group. The amount of phosphate that is associated with a protein is therefore precisely determined by the relative activities of the associated kinase and phosphatase. As much as 2% to 5% of the human genome is thought to encode protein kinases and phosphatases.
The most common amino acids to be phosphorylated on eukaryotic proteins (proteins found in all organisms except bacteria) are serine, threonine, and tyrosine.
At the level of a single protein, the binding of a negatively charged phosphate group can lead to changes in the structure of a protein, which alter the way that it functions. If the targeted protein is an enzyme, phosphorylation and dephosphorylation can impact its enzymatic activity, essentially acting like a switch, turning it on and off in a regulated manner.
Another outcome of structural changes to the phosphorylated protein is the facilitation of binding to a partner protein. In this way, phosphorylation can regulate protein-protein interactions. The phosphorylation of a protein can also target it for degradation and removal from the cell by the ubiquitin-proteasome system.
Protein phosphorylation also has a vital role in intracellular signal transduction. Many of the proteins that make up a signaling pathway are kinases, from the tyrosine kinase receptors at the cell surface to downstream effector proteins, many of which are serine/threonine kinases.
In a nutshell, ligand binding at the cell surface establishes a phosphorylation cascade, with the phosphorylation and activation of 1 protein stimulating the phosphorylation of another, subsequently amplifying a signal and transmitting it through the cell. The signal continues to propagate until it is switched off by the action of a phosphatase.
In addition to proteins, other kinds of molecules can also be phosphorylated. In particular, the phosphorylation of phosphoinositide lipids, such as phosphatidylinositol-4,5-bisphosphate (PIP2), at various positions on their inositol ring, also plays a key role in signal transduction.
Phosphorylation plays a vital role in regulating many intracellular processes such as growth, proliferation, and cell division. Thus, any perturbations in the phosphorylation process are likely to drive many of the hallmarks of cancer, such as unchecked cell growth and proliferation. Indeed, mutations in kinases and phosphatases are frequently implicated in a number of different cancers, and many of the genes encoding for these proteins are oncogenes or tumor suppressors.
Overexpression or mutations that lead to constitutive activation of phosphorylation machinery will inevitably disrupt its delicate balance in the cell, driving the inappropriate activation or deactivation of the cellular processes it controls.
The key function of protein kinases in signal transduction has made them an attractive target for cancer therapeutics.
The key function of protein kinases in signal transduction has made them an extremely attractive target for therapeutic intervention in cancer. Protein kinases represent as much as 30% of all protein targets under investigation by pharmaceutical companies. Targeting tyrosine kinases in particular is a popular approach, and ground-breaking advances have been made in recent decades with the introduction of this class of agents.
They include drugs such as trastuzumab (Herceptin, Genentech), a monoclonal antibody designed to block the function of the HER2 receptor tyrosine kinase, which revolutionized the treatment of breast cancer, and erlotinib (Tarceva, Genentech), a receptor tyrosine kinase inhibitor, that targets the epidermal growth factor receptor (EGFR) and is approved by the FDA for treatment of patients with non-small cell lung cancer.
More recently, the serine/threonine kinases have also emerged as strong candidates and around one-third of all kinase inhibitors currently in development target serine/threonine kinases. Most advanced among this class of drugs are those targeting the mammalian target of rapamycin (mTOR): everolimus (Afinitor, Novartis) and temsirolimus (Torisel, Pfizer), both approved for the treatment of renal cell carcinoma.
A number of inhibitors targeting AKT are also in the pipeline, including perifosine (Keryx Biopharmaceuticals, Inc), and there is a substantial amount of interest in drugs targeting MEK, a critical kinase at the junction of several biological pathways that regulate cell proliferation, survival, migration, and differentiation, including AZD6244 (selumetinib, AstraZeneca).
Serine/threonine kinases involved in cell cycle regulation are also heavily investigated, such as the aurora kinases and the cyclin-dependent kinases. Among them are AZD1152 (AstraZeneca) and HMR-1275 (alvocidib; sanofi -aventis), which are in various stages of clinical development.
Given the pivotal roles of phosphorylation in the cellular environment, and the fact that the overwhelming majority of phosphorylation sites remain uncharacterized, there is a constant effort by researchers to better understand the role of phosphorylation and to develop novel, highly sensitive, and sophisticated phosphorylation identification techniques.
The gold-standard method for measuring protein phosphorylation levels is an in vitro kinase assay, in which radioactive ATP is used. When a phosphorylation event occurs, the radioactive inorganic phosphate (32P) in ATP will be transferred to the phosphorylated protein; thus, following the radioactivity allows researchers to establish when proteins are phosphorylated. The development of specific antibodies against phosphorylated forms of proteins led to the use of immunoassays to examine phosphorylation patterns of individual proteins.
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More recently, researchers have begun to make use of increasingly sophisticated mass spectrometry and nuclear magnetic resonance imaging methods to try to decipher global patterns of phosphorylation within the cell in response to certain stimuli or therapeutic agents. The potential for using the phosphorylation status of key signaling proteins as a diagnostic biomarker, predictor of response, or prognostic indicator is an area of intense interest, reflected in a number of presentations at this year’s American Society of Clinical Oncology (ASCO) meeting.
Studies are examining the utility of phosphorylated forms of epidermal growth factor receptor and HER2 as both biomarkers of response to tyrosine kinase inhibitors and as prognostic indictors. For example, 1 study found that a high level of expression of phosphorylated HER2 in patients with HER2- positive breast cancer is associated with lower 5-year disease-free survival.
Furthermore, researchers are beginning to exploit binding domains within signaling proteins that specifically recognize and bind to other phosphorylated proteins. Using the Src homology 2 (SH2) -binding domain, which binds to phosphorylated tyrosines, researchers have been able to analyze the global state of tyrosine phosphorylation in different human cancer cell lines to see how it differs from normal cells.
Jane de Lartigue, PhD, is a freelance medical writer and editor based in the United Kingdom.