Contributed by Mike Browning and Amy Archuleta
For proteins, form IS function.
Proteins do what they do because of their shape. Structural proteins function constantly to underpin cellular structure. Other proteins mediate biological functions (e.g. cell division) that must be regulated and turned off and on as the cell requires. Protein phosphorylation is the primary cellular mechanism for regulating a protein’s structure and hence its activity. Protein phosphorylation occurs when a protein kinase transfers phosphate from ATP to serine, threonine, or tyrosine residues on the target protein. The addition of the negatively charged phosphate group changes the shape, and hence the function, of the target protein.
Phosphoproteins are considered to be among the most important proteins in the body.
Phosphoproteins are the proteins that regulate almost all cell processes, from cell division in cancer to neuronal signal transduction in learning and memory. Since phosphoproteins regulate virtually every important cellular function, we like to say that “phosphoproteins are the verbs of the proteomic language.” The ability to assay the state of phosphorylation of specific proteins is of great utility in the quest to establish the function of a given protein and how that activity is influenced by cellular signals. Such assays are also critical for the identification of drugs that can influence the phosphorylation and hence the function of specific proteins.
A method was needed to track the activity of a protein, not simply its level of expression.
To visualize the importance of such a tool, imagine an FBI agent sitting in a car surveilling a suspect inside a house. The agent may not be able to see what room the suspect is in or what they are doing. Contrast that with an agent with a video camera inside the house. Similarly, a phosphospecific antibody doesn’t just indicate that a protein is present, it shows whether the protein is active.
The history of phosphoprotein detection started radioactively.
Unfortunately, visualizing phosphoproteins in early studies required radiolabeling the phosphate group with 32P. This was labor intensive: literally thousands of proteins are being phosphorylated (and incorporating 32P) and thus immunoprecipitation is required to separate the protein of interest from all the other labeled proteins. However, even when immunoprecipitation is performed and the protein of interest is isolated, it is not possible to determine which site on the protein has been phosphorylated. This is critical as some sites on a protein can turn it on while others can turn it off. Moreover, 32P studies are very difficult to quantitate as metabolic changes alone can alter the 32P level of labeling. Lastly, studies of in vivo phosphorylation are virtually impossible due to the inability to label with 32P in vivo.
The development of phosphospecific antibodies started decades ago with antibodies against phosphotyrosine.
The first report of the production of phosphorylation-dependent antibodies appeared in 1981, when antibodies that could detect phosphotyrosine-containing proteins were produced by injecting benzyl phosphonate into rabbits (1). These antibodies became key reagents in cancer research but detected phosphotyrosine on many proteins. The key downside of such antibodies is not only that they detect phosphorylation of many different proteins but also that, similarly to the 32P studies, they often detect multiple phosphorylation sites on the same protein. In essence such antibodies show that phosphorylation is occurring but can’t identify which proteins and which sites on the protein were phosphorylated. A number of attempts were also made to generate antibodies that detected phosphoserine or phosphothreonine but these were by-and-large not very successful.
Using full-length proteins as antigens was more targeted, but still proved problematic.
Shortly after this initial work, Nairn and colleagues described the production of an antibody specific for the phosphorylated form of G-substrate, a protein localized to cerebellar Purkinje cells and phosphorylated by cGMP-dependent protein kinase (2). The antibody was prepared by immunization of rabbits with the purified G-substrate phosphoprotein that had been phosphorylated in vitro. Despite this initial success, other attempts to produce phosphospecific antisera by immunization with the phospho-form of intact proteins were not very successful, probably because of two significant factors. First, many phosphorylated proteins are believed to undergo rapid dephosphorylation during immunization, regardless of the route of injection, leading to the loss of the desired phospho-epitope. Second, holoproteins generally contain multiple immunogenic epitopes. Since the phosphosite is only a very short piece of this long protein, there is a low probability the phosphosite of interest will be recognized by antibodies made against full-length protein antigens.
A more direct approach for generating site-specific phospho antibodies utilizes phosphorylated forms of synthetic peptides.
Short peptides made of the 15-20 amino acids immediately surrounding the phosphorylated amino acid of interest focus the immune response to just that specific phosphosite. Moreover, these short peptides are generally resistant to dephosphorylation. However this is not absolute as some of the peptides do become dephosphorylated. This leads to the generation of antibodies to the non-phosphorylated peptide as well. Consequently it is essential that the serum collected after these peptide immunizations be purified to separate the phospho peptide antibodies from the antibodies that recognize the dephopsho peptide.
Both the phospho and dephospho-peptides are of great utility in the purification of phosphospecific antibodies.
A general protocol for the production of phosphorylation state-specific antibodies was developed by one of our co-founders: Andy Czernik and his colleagues (3). Through sequential chromatography, phospho antibodies are first positively selected away from dephospho antibodies by passing the sera over a column bound with the phosphorylated peptide used as antigen. This step is followed by negative selection over a column bound with the dephosphorylated form of the peptide. Details of this purification procedure can be found on our website.
Today’s phosphospecific antibodies only bind to target proteins when the protein is in its phosphorylated state.
Through the development of this revolutionary immunochemical technique, it is now possible to develop phosphospecific antibodies that can be used to determine the level of phosphorylation of a specific site on a particular protein. They are much easier to work with since the antibody labels ONLY the protein of interest. IP is not necessary to separate out other proteins and there is no need for using 32P labeling. The antibodies thus can be used in both WB and IHC to show the relative intensity of phosphorylation of the specific site on the target protein. Moreover, when this antibody is used in IHC, it is possible to identify the specific cells in which the protein has been activated. We will discuss using phosphospecific antibodies in IHC in our blog.
Understanding of phosphoprotein function has led to great advancement in cancer treatments.
Phosphospecific antibodies have revolutionized the study of protein phosphorylation and greatly aided the development of a host of new drugs that target phosphoproteins to treat disease states. It is in cancer treatment that drugs affecting phosphorylation are most widely used and researched. The most common phosphorylation related drugs are small molecule kinase inhibitors. Such inhibitors account for nearly a quarter of current drug discovery and development efforts. Inhibition of distinct kinase signaling pathways can be less cytotoxic to non-cancerous cells thus affecting selective killing of cancer cells with less toxicity (4).
Beyond Western blot: An unexplored opportunity for IHC and IF.
Phosphospecific antibodies are very widely used in Western blot analyses of phosphoproteins. However, there has been considerably less use of these antibodies in the wonderful world of immunohistochemistry. The lack of studies using IHC/IF is unfortunate given the pronounced detail about the cellular and subcellular localization of phosphoproteins such studies could provide. At PhosphoSolutions, we have gone to great lengths to validate many of our phospho-antibodies for IHC/IF. In addition, we have written a chapter discussing the use of these antibodies in IHC/IF. In a subsequent blog we will discuss best practices for the use of phospho-antibodies in IHC/IF.
A final personal note from our founder, Mike Browning
When I started graduate school many years ago, only 2 protein kinases were known. Today we know that the human genome encodes for more than 500 kinases. Nevertheless, it was incredibly exciting to discover this fascinating world where proteins could be turned off and on again like a light bulb. When I learned of this process it was like a light bulb went of in my head. I was studying the way brain cell signaling was plastic, i.e. it could be turned on and off or even up and down like the volume control on a stereo. And here right in front of me was the perfect molecular mechanism to underpin brain plasticity: Plastic proteins! I was hooked, and have been ever since. Thank goodness for phosphoantibodies or I would probably be radioactive with 32P by now.
- Ross AH, Baltimore D, Eisen HN (1981) Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature. 294(5842):654-6.
- Nairn AC, Detre JA, Casnellie JE, Greengard P (1982) Serum antibodies that distinguish between the phospho- and dephospho-forms of a phosphoprotein. Nature (London) 299:734-736.
- Czernik AJ, Girault J-A, Nairn AC, Chen J, Snyder G, Kebabian J, Greengard P (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201:264-283.
- Bhullar KS, Lagarón NO, McGowan EM, Parmar I, Jha A, Hubbard BP, Rupasinghe HPV. Kinase-targeted cancer therapies: progress, challenges and future directions. Mol Cancer. 2018 Feb 19;17(1):48. doi: 10.1186/s12943-018-0804-2. PMID: 29455673; PMCID: PMC5817855.)
Read educational articles and Phospho news on our blog!
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- If we attempt to make an antibody and it doesn’t work, we throw it away. We never sell an antibody that we can’t get to work in our hands.
- Our commitment to strict validation standards ensures a clean signal in endogenous protein and verifies phosphospecificity for relevant products.
- We are dedicated to reproducibility. Look for our “Pooled Serum” icon. Each antibody with this icon is purified from its own pool of serum to ensure lot-to-lot consistency.
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