Protein Phosphorylation
Phosphorylation is the main form of reversible covalent modification of proteins for their regulation. This is catalyzed by a large family of enzymes called protein kinases. The dephosphorylation of phosphoproteins is carried out by another group of enzymes called protein phosphatases. The study of these enzymes and their phosphoprotein targets (also known as substrates) is having an enormous impact on our understanding of how cells are controlled and human health care delivery.
Phosphorylation acts as a molecular switch to directly turn on or off the functions of proteins, or it may more subtly regulate their functions, for example by controlling their locations inside of cells, interactions with other proteins, or their degradation by proteases. The phosphate group that is transferred onto proteins by protein kinases is removed from the high energy compound adenosine-triphosphate (ATP), which fuels most chemical reactions in the body. Research conducted by Kinexus is supporting the novel hypothesis that the predominant function of hyperphosphorylation is to mediate the destruction of proteins.
It is now known that more than 80% of the different proteins encoded by genes (over 17,000) are subjected to protein phosphorylation, and that the vast majority of these are phosphorylated at multiple sites per protein. Phosphorylation is found most commonly on specific serine and threonine amino acid residues in proteins, but it also occurs on tyrosine and other amino acid residues (histidine, aspartic acid, glutamic acid) as well. In most cells, about 90% of the protein phosphorylation appears on serine, 10% on threonine and less than ~0.05% on tyrosine residues. Therefore, protein-tyrosine phosphorylation levels appear to be about 2000-times lower than protein-serine phosphorylation levels and 200-times lower than protein-threonine phosphorylation levels. In cells like activated platelets, tyrosine phosphorylation can be orders of magnitude higher.
From random mass spectrometry analyses of phosphosites in proteins, it appears that around 76% of the phosphorylation sites feature serine as the modified amino acid residue, whereas threonine and tyrosine, respectively, account for about 20% and 4% of the number of phosphosites in proteins. This difference in total amount of protein-tyrosine phosphorylation and the frequency of occurrence of tyrosine phosphorylation sites in proteins appears to reflect higher levels of tyrosine phosphorylation sites in lower abundance proteins such as protein kinases.
Kinexus has now performed a careful analysis of over 180,000 known human phosphorylation sites. Information on most of these phosphosites is available through our on-line PhosphoNET Knowledgebase. For those proteins where an effect on protein function has been established (less than 5% of the known phosphosites), in 70% of these phosphorylation promotes the protein’s function, whereas 30% of these phosphosites appear to be inhibitory. Based on the number of known tyrosine phosphorylation sites and their relative occurrence, we conclude that there is likely to be over a million human phosphorylation sites. We believe that the vast majority of the 21,000 proteins encoded by the human genome are in fact phosphorylated. Moreover, the average protein would seem to be phosphorylatable on more than 40 amino acid residues. However, only a smaller number of these sites are strongly phosphorylated based on their frequency of detection in mass spectrometry studies. We hypothesize that following initial phosphorylation of proteins to directly regulate their activities, they undergo hyperphosphoryation and become tagged for degradation.
Phosphorylation acts as a molecular switch to directly turn on or off the functions of proteins, or it may more subtly regulate their functions, for example by controlling their locations inside of cells, interactions with other proteins, or their degradation by proteases. The phosphate group that is transferred onto proteins by protein kinases is removed from the high energy compound adenosine-triphosphate (ATP), which fuels most chemical reactions in the body. Research conducted by Kinexus is supporting the novel hypothesis that the predominant function of hyperphosphorylation is to mediate the destruction of proteins.
It is now known that more than 80% of the different proteins encoded by genes (over 17,000) are subjected to protein phosphorylation, and that the vast majority of these are phosphorylated at multiple sites per protein. Phosphorylation is found most commonly on specific serine and threonine amino acid residues in proteins, but it also occurs on tyrosine and other amino acid residues (histidine, aspartic acid, glutamic acid) as well. In most cells, about 90% of the protein phosphorylation appears on serine, 10% on threonine and less than ~0.05% on tyrosine residues. Therefore, protein-tyrosine phosphorylation levels appear to be about 2000-times lower than protein-serine phosphorylation levels and 200-times lower than protein-threonine phosphorylation levels. In cells like activated platelets, tyrosine phosphorylation can be orders of magnitude higher.
From random mass spectrometry analyses of phosphosites in proteins, it appears that around 76% of the phosphorylation sites feature serine as the modified amino acid residue, whereas threonine and tyrosine, respectively, account for about 20% and 4% of the number of phosphosites in proteins. This difference in total amount of protein-tyrosine phosphorylation and the frequency of occurrence of tyrosine phosphorylation sites in proteins appears to reflect higher levels of tyrosine phosphorylation sites in lower abundance proteins such as protein kinases.
Kinexus has now performed a careful analysis of over 180,000 known human phosphorylation sites. Information on most of these phosphosites is available through our on-line PhosphoNET Knowledgebase. For those proteins where an effect on protein function has been established (less than 5% of the known phosphosites), in 70% of these phosphorylation promotes the protein’s function, whereas 30% of these phosphosites appear to be inhibitory. Based on the number of known tyrosine phosphorylation sites and their relative occurrence, we conclude that there is likely to be over a million human phosphorylation sites. We believe that the vast majority of the 21,000 proteins encoded by the human genome are in fact phosphorylated. Moreover, the average protein would seem to be phosphorylatable on more than 40 amino acid residues. However, only a smaller number of these sites are strongly phosphorylated based on their frequency of detection in mass spectrometry studies. We hypothesize that following initial phosphorylation of proteins to directly regulate their activities, they undergo hyperphosphoryation and become tagged for degradation.
Our research indicates that proteins can be typically phosphorylated at once every 17 amino acids, although in reality phosphorylation appears to occur in dense clusters on neighbouring amino acid residues. Since at least one kinase and one phosphatase would be the minimum number of enzymes targeting each phosphorylation site, this would indicate at least 2 million possible connections in phosphorylation signalling networks. However, most phosphorylation sites are likely to be each targeted by many multiple broad-specificity kinases and phosphatases. Therefore, it is more likely that over 10 million possible connections exist within these regulatory networks. Kinexus is working to successfully predict most of these connections and define the most critically important.
While the amino acid sequences of proteins found in humans and other mammals are highly similar (in the order of 80 to 99% identical), there are profound differences in their phosphoproteomes. For example, the phosphoproteomes of men and mice appear to be around 25% different. This means that their cell signalling maps have to be similarly different. Moreover, less than 8% of the human phosphorylation sites are conserved in the fruit fly and nematode worm, and less than 3% in yeast - Therefore, despite their popularity, these experimental model systems appear to have very little relevance to the human condition. For this reason, most of Kinexus’ internal research programs have been focused on the analyses of human cells and tissues, and we would strongly encourage the biomedical research community to follow suite.
The unique complement of phosphorylated proteins within a cell defines its phosphoproteome. The phosphoproteomes of cells are more dynamic than their proteomes and even more reflective their health and activation status. The phenotype of a cell is a consequence of its levels of active proteins. In our proteomics analyses, Kinexus has often noted an inverse correlation between the total amounts and phosphorylated levels of specific proteins. 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.
While the amino acid sequences of proteins found in humans and other mammals are highly similar (in the order of 80 to 99% identical), there are profound differences in their phosphoproteomes. For example, the phosphoproteomes of men and mice appear to be around 25% different. This means that their cell signalling maps have to be similarly different. Moreover, less than 8% of the human phosphorylation sites are conserved in the fruit fly and nematode worm, and less than 3% in yeast - Therefore, despite their popularity, these experimental model systems appear to have very little relevance to the human condition. For this reason, most of Kinexus’ internal research programs have been focused on the analyses of human cells and tissues, and we would strongly encourage the biomedical research community to follow suite.
The unique complement of phosphorylated proteins within a cell defines its phosphoproteome. The phosphoproteomes of cells are more dynamic than their proteomes and even more reflective their health and activation status. The phenotype of a cell is a consequence of its levels of active proteins. In our proteomics analyses, Kinexus has often noted an inverse correlation between the total amounts and phosphorylated levels of specific proteins. 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.