Protein Phosphorylation -- (1) (from Wikipedia, the free encyclopedia)
Contents
1.4 Protein phosphorylation sites
Methods for Detecting Protein Phosphorylation
Introduction
Protein kinases transfer phosphate groups from ATP to serine, threonine, or tyrosine residues on protein peptide substrates, directly affecting the activity and function of the target. Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation.1,2 This crucial post-translational modification regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and neuronal communication. In addition, abnormal phosphorylation events are implicated in many disease states. When assessing phosphorylation, the method of choice may vary depending on many factors including the specific question being asked and availability of specialized equipment or reagents. This review provides a brief description of several methodologies currently used and addresses some of the benefits and drawbacks associated with each.
Kinase Activity Assays
Protein kinases are often common elements in multiple signaling networks influencing numerous downstream effectors responsible for a biological response. Thus, assessing the activity of a single specific kinase may provide valuable information on parallel pathways.3 Kinase activity within a biological sample is commonly measured in vitro by incubating the immunoprecipitated kinase with an exogenous substrate in the presence of ATP. Measurement of the phosphorylated substrate can be assessed by several reporter systems including colorimetric, radioactive, or fluorometric detection.4 Although information can be obtained regarding the actions of a specific kinase, assessing enzyme activity in cellular extracts only provides a glimpse of the signaling landscape. Little is revealed about the proteins being modified, and in vitro activity assays do not address the role of potential endogenous phosphatase activity. Direct detection of phosphorylated proteins can provide a more detailed analysis of the cellular response to an external stimulus, as identification of a phosphopeptide provides information regarding the expression and the functional state of that protein.
Phospho-Specific Antibody Development
A
classical method of directly measuring protein phosphorylation involves the incubation of whole cells with radiolabeled 32P-orthophosphate,
the generation of cellular extracts, separation of proteins by SDS-PAGE, and exposure to film.2,5 This labor-intensive method requires
many multi-hour incubations and the use of radioisotopes. Other traditional methods include 2-dimensional gel electrophoresis, a technique
that assumes phosphorylation will alter the mobility and isoelectric point of the protein.
In light of these laborious methods, the
development of phosphorylation-dependent antibodies was a welcome event for researchers. In 1981, the first documented phospho-antibody
was produced in rabbits immunized with benzonyl phosphonate conjugated to keyhole limpet hemocyanin (KLH). This antibody broadly recognized
proteins containing phosphotyrosine.6 Ten years later, phosphorlyation state-specific (phospho-specific) antibodies were developed
by immunizing rabbits with synthetic phosphopeptides representing the amino acid sequence surrounding the phosphorylation site of
the target protein.7 The immune sera was applied to a peptide affinity column to generate a highly specific immunoreagent. The availability
of phospho-specific antibodies (Table 1) has opened the door for the improvement of traditional methods as well as the development
of new immunoassay techniques. The main caveat in utilizing phospho-specific antibodies in any technique is that successful detection
is dependent on the specificity and affinity of
Western Blot
The Western blot is the most common method used for assessing the phosphorylation state of a protein, and most cell biology laboratories possess the equipment necessary to perform these experiments. Following separation of the biological sample with SDS- PAGE and subsequent transfer to a membrane (usually PVDF or nitrocellulose), a phospho-specific antibody can be used to identify the protein of interest. The typical Western blot protocol eliminates the hazards and waste disposal requirements associated with the use of radioisotopes. Many phospho-specific antibodies are quite sensitive and can readily detect the phosphorylated protein in a routine sample (e.g., 10-30 ¦Ìg whole cell extract). Because the measured levels of a phospho-protein may change with treatment or through gel loading errors, researchers often utilize an antibody that detects the total level of the cognate protein (regardless of phosphorylation state) to determine the phosphorylated fraction relative to the total fraction and to serve as an internal loading control. Both chemiluminescent and colorimetric detection methods are common, and molecular weight markers are also generally used to provide information about protein mass.
Enzyme-Linked Immunosorbent
Assay (ELISA)
The ELISA has become a powerful method for measuring protein phosphorylation. ELISAs are more quantitative than Western blotting and show great utility in studies that modulate kinase activity and function. The format for this microplate-based assay typically utilizes a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein, either purified or as a component in a complex heterogeneous sample such as a cell lysate, is then bound to the antibody-coated plate. A detection antibody specific for the phosphorylation site to be analyzed is then added. These assays are typically designed using colorimetric or fluorometric detection. The intensity of the resulting signal is directly proportional to the concentration of phosphorylated protein present in the original sample. The phospho-specific ELISA technique confers several advantages over more traditional immunoblotting in the measurement of protein phosphorylation. First, results are easily quantifiable by utilizing a calibrated standard. Second, high specificity is possible due to the use of two antibodies specific for the target protein employed together in the sandwich format. Finally, the higher sensitivity often accomplished using ELISAs allows for smaller sample volumes and the detection of low abundance proteins. ELISAs generally provide an indirect measurement of kinase activity. However, variations in the technique described above use an immobilized capture antibody, substrate, and a phospho-substrate detection method for more direct measurements of kinase activity.
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