Protein phosphorylation is a common posttranslational modification in which a phosphate moiety is covalently bound to an amino acid. Phosphorylation occurs through the action of specific protein modifiers called kinases. The reverse reaction, dephosphorylation (removal of the phosphate group), is carried out by another set of enzymes called phosphatases. Phosphorylation of a protein is a dynamic, reversible event, dependent on the activity of both kinases and phosphatases. Due to its reversible nature, phosphorylation provides a quick, flexible, and sometimes transient, signal that cells use to quickly transmit information and respond to stimuli. Phosphorylation is the most common form of protein modification and is used in most signal transduction pathways controlling nearly every aspect of cellular life including:
- Cell cycle progression
- Cell growth and differentiation
- Cellular communication
Kinase biology is extremely complex. Approximately 500 different protein kinases and 200 protein phosphatases are predicted to be encoded by the human genome. Each kinase and phosphatase has a substrate specificity for a particular set of proteins. In addition, many proteins are phosphorylated on multiple sites and kinase recognition sites can overlap, leading to diverse and complex protein phosphorylation patterns.
Due to its complexity and transience, protein phosphorylation can be a challenge to study. However, several techniques have been developed to reliably study protein phosphorylation. Often, multiple techniques are used together to fully understand the phosphorylation status of a protein.
In eukaryotes, the most commonly phosphorylated amino acid residues are serine, threonine and tyrosine. To be phosphorylated, the residue must be presented in the context of a consensus site that can be recognized by the kinase. Consensus sites for many kinases have been determined empirically and published. Computer programs based on these consensus sites are used to predict potential phosphorylation sites in other proteins. However, kinases recognize 3-d structures that cannot always be easily predicted by computer software. Therefore, any predicted phosphorylation sites must be confirmed through other studies.
Radiolabeling of cells
Radiolabeling can be used to study phosphorylation in vivo. For these assays, live cells are incubated with 32P-orthophosphate which is taken up by the cells. Intracellular kinases then transfer the radiolabel to an appropriate protein substrate. Whole cell extracts can be separated by electrophoresis on an SDS-PAGE gel to look at total cellular protein phosphorylation. Alternatively, individual proteins can be immunoprecipitated prior to electrophoresis to examine phosphorylation of a specific protein. Often these assays are performed after stimulation of the cells to induce protein phosphorylation.
In vivo studies are useful in determining if a particular protein is phosphorylated without knowing the site of phosphorylation or the kinase involved. You can also use it in a pulse chase format or during different stimulus conditions to determine when phosphorylation occurs. Because proteins are exposed to both kinases and phosphatases within the cell, you detect the steady-state level of phosphorylation of the protein. However, phosphorylation can be difficult to detect if it is transient or only occurs on a few molecules.
Labeling of all phosphoproteins within a cell can also be achieved through the use of phospho-specific stains. Stains can be used directly in acrylamide gels or on membranes after transfer. Stains are quick and easy to use, and are compatible with downstream applications such as mass spectrometry or Western blotting.
Similar to whole cell radiolabeling, unless recombinant or immunoprecipitated proteins are run on the gel, stains do not allow you to visualize phosphorylation of a particular protein. In addition, phospho-specific stains are not highly sensitive, requiring low nanogram amounts of phosphorylated (not total) protein for detection.
If you have a suspect kinase and/or substrate in mind, you can do in vitro studies by incubating the kinase with its substrate in a tube. In vitro kinase assays can identify the kinase(s) responsible for phosphorylation of a protein and be used in competition assays. When used in conjunction with mutagenesis, they can also be used to confirm the specific residue that is phosphorylated.
Radiolabeling can be used in kinase studies with fractionated cell extracts, recombinant proteins or short peptide sequences. In this case, the target protein(s) is incubated with 32P-alpha-ATP and a specific kinase. Alternatively, many companies sell non-radioactive kinase assays in which kinase activity is measured by emittance of light, fluorescence or generation of color. Some of these kits detect byproducts of the reaction (e.g. ADP) rather than phosphorylation of the substrate. Many of these assays are quantitative and can be performed in a high through-put 96-wel format.
In vitro studies allow you to measure the potential activity of a kinase for a substrate. However, they do not reflect actual physiological levels of activity that may be controlled by accessibility of the phosphorylation site by the kinase. They also do not take into account the competing activity of the phosphatases or functionality of the kinases or substrates.
Antibodies have been developed that can detect phosphoserine, phosphothreonine or phosphotyrosine residues independent of the protein. In addition, highly specific antibodies that recognize a particular phosphorylated residue within an individual protein have also been developed (e.g. anti-phospho-MAP2 phosphorylated on serine 136). Phospho-specific antibodies can be used in an antibody-based assay such as Western blotting, enzyme-linked immunosorbent assays (ELISA), flow cytometry and immunohistochemistry/immunofluorescence. Western blot phosphorylation studies are highly quantitative when they are performed by multiplexing multiple fluorescent antibodies and the ELISA is useful when analyzing many samples.
Phospho-spcecific antibodies can be highly specific and sensitive and many are available from multiple manufacturers. However, highly specific antibodies can be costly. Additionally, keep in mind that phospho-specific antibodies recognize the phosphorylated residue and the surrounding epitope. If the phosphorylated residue is not presented within the correct epitope context, then it will not be recognized by the antibody. You may need to evaluate several different antibodies to detect phosphorylation of your protein of interest.
Mass spectrometry (mass spec) is being increasingly used to detect protein phosphorylation. Theoretically, mass spec analysis has the ability to detect and map all phosphorylated residues within a protein. In general, proteins are fragmented using proteases and protein masses and fragment ion masses are measured using tandem mass spectrometry. Phosphorylated residues are mapped by comparing the obtained data to predicted theoretical spectra that considers phosphorylation of every possible fragment.
In practice, mass spec has had significant limitations in the ability to detect all phosphorylated residues, particularly when the phosphorylated form of the protein is in low abundance compared to the unphosphorylated form. However, results can be improved with enrichment of the phosphorylated protein prior to analysis.
Helpful hints for phosphorylation studies
- Always use phosphatase inhibitors when preparing cell lysates.
- Keep samples cool to inhibit phosphatases.
- Add loading buffer to protein samples prior to storing to inactivate proteases and phosphatases.
- For antibody studies, do not use milk as a blocking agent. Milk contains phosphoproteins that can interact with the antibody.
- Do time courses to determine the time at which your protein of interest is maximally phosphorylated.
- Enrich or concentrate samples to increase the amount of phosphorylated protein in the sample.
- Confirm specificity of phospho-specific antibodies.
- Always use a positive and negative control.
Photo courtesy of Pink Sherbert Photography.