Which amino acids phosphorylated

If so, the protein should be treated with phosphatase during purification. If the protein is isolated by affinity chromatography, this can be achieved by treating the protein with lambda or calf intestinal phosphatase while it is still bound to the affinity beads. Even proteins produced in bacteria sometimes have undergone nonspecific phosphorylation and may need to be treated with phosphatase before being used as a substrate. Two reactions should be carried out: one containing kinase, and a reference reaction containing no kinase or a kinase-dead mutant.

If one is using protein labeled by SILAC, the reference and experimental reactions are combined after the reactions and should contain equal amounts of protein labeled with heavy or light isotope.

The combined reactions are analyzed directly by mass spectrometry after protease treatment to identify sites phosphorylated by the kinase. Alternatively, the combined reactions can be resolved by electrophoresis, and the band representing the protein of interest can be excised and analyzed by mass spectrometry to compare levels of phosphorylation, which can improve detection in some cases.

If one is not using SILAC, the kinase and control reactions can be independently labeled with mass tags, combined, and analyzed. As a complement to mass spectrometry analysis, phosphoamino acid analysis can be applied to the protein of interest isolated from 32 P-labeled cells or after in vitro phosphorylation by specific kinases Kamps and Sefton, For example, if phosphoamino acid analysis reveals that the protein of interest is phosphorylated on both serine and threonine residues in vivo and in vitro, but mass spectrometry analysis identifies only serine phosphorylation sites, it might be that potentially important modifications were missed because the corresponding threonine phosphopeptides could not be detected by mass spectrometry.

Similarly, two-dimensional phosphopeptide mapping can provide a sense of the complexity of phosphorylation, as well as a means of testing whether all important sites of phosphorylation have been identified after mutagenesis, without the need for complex mass spectrometry experiments Boyle et al. Classic phosphopeptide mapping also has the advantage that all the phosphopeptides are detected, in contrast to mass spectrometry.

To determine whether phosphorylation sites identified in vitro are relevant, it is important to show that they are also phosphorylated in vivo. A number of databases catalogue sites that have been found to be phosphorylated in vivo in large-scale surveys, so an easy first step is to determine whether sites identified in vitro have already been identified in vivo, while keeping in mind that large scale surveys provide low-sequence coverage and miss many sites for databases see PhosphoSitePlus, Phospho.

To map sites phosphorylated in vivo, one should first define physiological conditions under which a significant fraction of the protein is phosphorylated. This could involve treating cells with a stimulus that activates signaling or synchronizing cells in the cell cycle.

Another approach is to manipulate the signaling pathway genetically such that the kinase of interest is hyperactivated. An ideal situation is when one can purify the substrate protein from control cells, cells in which the relevant kinase is hyperactivated, and cells in which the relevant kinase has been inactivated, which allows one to determine which sites depend on the kinase of interest in vivo.

Once the appropriate conditions have been defined, the substrate protein must be purified under conditions that preserve phosphorylation and yield sufficient amounts of protein. Affinity purification methods that allow specific release of the purified protein will produce the best results. For example, proteins tagged with hemagglutinin or FLAG can be purified using antibody beads and eluted with an excess of peptide Ho et al.

Purification of proteins using antibodies raised against the protein of interest can be problematic because elution from the antibody requires harsh conditions that also release large amounts of antibody from the beads, complicating the analysis, although this problem can be circumvented by cross-linking the antibodies to beads. Nonspecific elutions also generate a higher background of contaminant proteins that can interfere with target peptide detection. To preserve phosphorylation, purification should be carried out in buffers that contain high concentrations of salt and phosphatase inhibitors.

High salt concentrations help inhibit phosphatases and reduce nonspecific binding, whereas phosphatase inhibitors minimize dephosphorylation during purification. Mass spectrometry analysis will yield a list of identified sites. Researchers should be aware that the data will contain incorrect matches. As discussed earlier, in many cases site assignments cannot always be resolved to a single residue.

In some cases, for example, where the study is focused on a kinase with a known consensus motif, local sequence can be used to guide the choice of sites to pursue for further validation. However, in most cases, all possible sites on each peptide must be considered. Sites that are phosphorylated both in vivo and in vitro have high confidence of being relevant sites.

Sites that change in occupancy in response to changes in relevant upstream signals are also high-confidence sites. Another consideration that can enhance confidence that correct identification has been made is the conservation of the phosphorylation site s throughout evolution. Although proteins phosphorylated at multiple sites within unstructured regions may not show evolutionary conservation of phosphorylation sites, within folded domains, phosphorylation sites are often conserved Landry et al.

Phosphorylation-site mapping screens are almost never saturating. Failure to observe a site is insufficient evidence to conclude that the site is not phosphorylated in the cell.

Many factors, including length, hydrophobicity, and charge, affect the chromatographic properties and ionization efficiencies and thus the ease of detection of different peptides. Phospho and nonphospho forms of the same peptide can have very different signal intensities. Thus even the observation of an unphosphorylated peptide is no guarantee that the correlate phosphopeptide is easily detectable. Despite these caveats, the unphosphorylated peptide sequence coverage still provides some indication of the depth of analysis.

Lower coverage decreases confidence that all sites have been identified and often indicates that more protein or an additional protease is needed to generate peptides for the analysis. For quantitative experiments, the data will include abundance ratios for each phosphopeptide along with signal intensities, often recorded as a signal-to-noise ratio.

Unlike protein-level analysis, in which multiple quantified peptides are often observed, phosphopeptides are more frequently detected and quantified only once. There is a strong correlation between signal strength and reproducibility. When selecting sites for further study, investigators should pay close attention to the number of PSMs and the signal strength for peptides harboring each site. It should also be noted that compiling ratios at the site level from multiple peptide measurements is not always trivial.

Simply calculating averages or medians of all peptides containing a given site might not reveal the full complexity of cellular phosphorylation patterns. Singly and doubly phosphorylated forms of a peptide might be present at different levels.

One must also not forget that changes in total protein level are not reflected in the phosphopeptide ratios. Wherever possible, separate protein-level measurements made from unmodified peptides should be performed and used to normalize phosphopeptide ratios.

After identification of all phosphorylation sites possible and their assignment to likely protein kinases, the next step is to mutate the sites so that their biological significance can be ascertained.

Typically, serine and threonine phosphosites are mutated to alanine or valine for threonine , and tyrosine phosphorylation sites are mutated to phenylalanine. Because mass spectrometry can miss sites, it is important to verify that most or all key sites have been identified and mutated. However, this does not exclude the possibility that some sites have been missed that do not cause a shift.

Thus a more rigorous approach is to show that the mutant protein fails to incorporate 32 P in a reconstituted in vitro system or that the relevant phosphopeptides identified by in vivo, 32 P-labeled, two-dimensional phosphopeptide mapping disappear. It can sometimes also be informative to switch serine for threonine residues or vice versa. Many protein kinases do not distinguish serine from threonine, and if the site is targeted in vivo, the protein's gel shift should not be lost with this switch, and yet a change in phosphoamino acid content of the corresponding peptide can be readily identified, which allows one to directly verify that the relevant phosphorylation site has been identified.

If a phosphorylation site mutant causes a loss of function, there can be the concern that it causes nonspecific damage to the protein. The vast majority of phosphorylation sites occur in regions of proteins that are predicted to be disordered, so it is unlikely that phosphorylation-site mutants disrupt protein structure Iakoucheva et al. In addition, a number of criteria can be used to help rule out this possibility.

For example, if normal levels of the protein are expressed in vivo, it is likely that the protein undergoes normal folding, since proteins that cannot fold correctly are destroyed. Another helpful test is to determine whether the phosphorylation-site mutant retains a subset of normal functions, which would indicate that the mutants affect specific functions of the protein. If the protein shows normal localization, it clearly retains key functions. In this approach, serine and threonine are typically mutated to aspartic or glutamic acid residues, whereas tyrosine is substituted with glutamic acid.

This approach has two significant shortcomings. First, if the phosphorylation site serves as a recognition signal for an adaptor protein i. Neighboring pairs of aspartic or glutamatic acid side chains can overcome the charge differential and may act as better phosphomimetics Strickfaden et al. However, the size of the ionic shell produced by a phosphate group is also different, and so the overall chemical environment created by phosphorylation is very different from that of negatively charged amino acids Hunter, It is therefore not surprising that phosphomimetic mutations often fail to reproduce the changes to a protein caused by phosphorylation.

As a result of these two limitations, the behavior of phosphomimetic mutations can be uninterpretable. Of course, there are examples in which phosphomimetic substitutions have been highly informative. The constitutive activation of MEK kinases by a phosphomimetic mutation is an excellent example McKay and Morrison, Rigorous, high-confidence mapping results come from an approach that combines information obtained by mapping sites on protein phosphorylated in vivo and protein phosphorylated in vitro by purified kinase.

Because many proteins are phosphorylated by multiple kinases, care must be taken to ensure that sites identified by mapping can be unambiguously assigned to the relevant kinase. Obtaining high sequence coverage requires a minimum of 2—10 pmol of purified phosphorylated protein, which corresponds to — ng of a kDa protein.

More protein is better. Even under the best conditions, mass spectrometry can miss phosphorylation sites because the corresponding peptides are lost during sample preparation or are not detected by mass spectrometry.

Before beginning a mapping project, it is important to consider whether good in vivo and in vitro assays are available to determine whether all key sites have been mutated.

One should also consider whether good assays are available to analyze the functions or phenotypes of phosphorylation-site mutants. This article is distributed by The American Society for Cell Biology under license from the author s. Two months after publication it is available to the public under an Attribution—Noncommercial—Share Alike 3. Molecular Biology of the Cell Vol. Kathleen L. Steven P. Douglas R. View PDF.

Add to favorites Download Citations Track Citations. Abstract A mechanistic understanding of signaling networks requires identification and analysis of phosphorylation sites. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

Nat Biotechnol 24 , Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4 , Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates.

Methods Enzymol , Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. Cell 78 , Diverse mitogenic agents induce the phosphorylation of two related 42,dalton proteins on tyrosine in quiescent chick cells. Mol Cell Biol 4 , Bioinformatics 20 , Scaffold proteins in MAP kinase signaling: more than simple passive activating platforms.

It appears that many regulatory proteins may reside in a cell in reserve in an inactive site. Upon their stimulation by phosphorylation, these proteins may then be hyperphosphorylated for their destruction. If this is a general phenomena, then tracking phosphoprotein levels will be much more insightful than monitoring the total levels of these proteins. With over one million human phosphosites, Kinexus believes that the phosphoproteome represents a largely untapped and important source of biomarkers for disease diagnosis.

It is our opinion that the sequencing of the human phosphoproteome is the next logical major human health initiative after the sequencing of the human genome.

The ability of some existing laboratories to identify hundreds of phosphosite a day with current mass spectrometry technology makes this an endeavor that could be accomplished within 5 years.

Kinexus has developed algorithms for the prediction of the identities of the one million human phosphosites and the protein kinases that are most likely responsible for their phosphorylation. The fruits of these bioinformatics efforts have been posted on-line in our PhosphoNET website.

Click here for links to other websites for more information about phosphorylation. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated. Jagarlamudi KK, Hansson LO and Eriksson S: Breast and prostate cancer patients differ significantly in their serum thymidine kinase 1 TK1 specific activities compared with those hematological malignancies and blood donors: implications of using serum TK1 as a biomarker.

BMC Cancer. Sci Rep. Tumour Biol. Genes Cancer. N Engl J Med. Nat Genet. Adv Exp Med Biol. Cancer Biol Ther. Ahmadian MR: Prospects for anti-ras drugs.

Br J Haematol. Goodsell DS: The molecular perspective: the ras oncogene. Sawyers CL: Shifting paradigms: the seeds of oncogene addiction. Hainaut P and Plymoth A: Targeting the hallmarks of cancer: towards a rational approach to next-generation cancer therapy.

Curr Opin Oncol. Clin Pharmacol Ther. Druker BJ: Imatinib mesylate in the treatment of chronic myeloid leukaemia. Expert Opin Pharmacother. Mol Clin Oncol. Clin Lung Cancer. J Natl Cancer Inst. Hasskarl J: Sorafenib: targeting multiple tyrosine kinases in cancer. Recent Results Cancer Res. Am J Physiol Endocrinol Metab.

Oncol Rep. Eur J Clin Pharmacol. Axelsson J, Rippe A and Rippe B: mTOR inhibition with temsirolimus causes acute increases in glomerular permeability, but inhibits the dynamic permeability actions of puromycin aminonucleoside. Am J Physiol Renal Physiol. Cutillas PR: Role of phosphoproteomics in the development of personalized cancer therapies.

Proteomics Clin Appl. Cancer Discov. Sci Transl Med. Murray BW and Miller N: Durability of kinase-directed therapies - a network perspective on response and resistance. Mol Cancer Ther.

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International Journal of Molecular Medicine. This article is mentioned in:. Introduction Protein phosphorylation is one of the most common and important post-translational modifications PTMs 1 , 2. Figure 1 Phospho-signaling networks.

Table I Subfamilies of protein kinases. Protein kinase family Origin of the name Description Refs. They are important for expression of various genes because after activation, CAMKs phosphorylate several transcription factors.

Abnormalities in MAP kinase cascades are tightly linked to oncogenic transformation 23 GSK3, initially described as a key enzyme involved in glycogen metabolism, is now known to regulate a diverse array of functions. TKs are cell surface receptors RTKs and many of the others function close to the surface of the cell 19 TKL T yrosine k inase- l ike Tyrosine kinase-like kinases are serine-threonine protein kinases named so because of their close sequence similarity to tyrosine kinases.

Related Articles. Tweets by IJMMedicine. Follow IJMMedicine. This site uses cookies. Spandidos Publications style. Int J Mol Med , Ardito, F. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy Review. International Journal of Molecular Medicine, 40, International Journal of Molecular Medicine International Journal of Molecular Medicine 40, no.