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Comparison of peptide array substrate phosphorylation of c-Raf and mitogen activated protein kinase kinase kinase 8.

Parikh K, Diks SH, Tuynman JH, Verhaar A, Löwenberg M, Hommes DW, Joore J, Pandey A, Peppelenbosch MP - PLoS ONE (2009)

Bottom Line: Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology.The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes.Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Section Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. k.parikh@med.umcg.nl

ABSTRACT
Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

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Analysis of phosphorylation of 1176 peptide array by c-Raf.A. In vitro phosphorylation of MEK by c-Raf. The capacity of purified c-Raf for in vitro phosphorylation studies was examined by incubating purified c-Raf with MEK and detected using MEKSer218/222/MEK2Ser222/226 antibodies. B. c-Raf phosphorylation of 1176 peptide array. Phosphorylation of the 1176 peptide array, spotted in duplicate, on incubation with c-Raf and 33P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation. Further analysis was carried out to determine whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation. C. Consensus sequence of c-Raf substrates using 1176 array design. Consensus sequence determined for c-Raf substrates on analysis of peptide array data shows a strong selection for both hydrophobic and basic residues at the −3 position. A strong preference for leucine is seen at the −2 position. Proline and arginine are strongly preferred at the −1 position. An examination of the amino acid preferences C-terminal to the fixed phosphorylated residue reveals a bias towards aspargine compared to other residues at the +1 position. Also, acyclic and hydrophobic amino acids are seen at the +1 position with no preference for any particular group of amino acid at the +2 position. The +3 position shows a strong preference for basic residues.
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pone-0006440-g002: Analysis of phosphorylation of 1176 peptide array by c-Raf.A. In vitro phosphorylation of MEK by c-Raf. The capacity of purified c-Raf for in vitro phosphorylation studies was examined by incubating purified c-Raf with MEK and detected using MEKSer218/222/MEK2Ser222/226 antibodies. B. c-Raf phosphorylation of 1176 peptide array. Phosphorylation of the 1176 peptide array, spotted in duplicate, on incubation with c-Raf and 33P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation. Further analysis was carried out to determine whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation. C. Consensus sequence of c-Raf substrates using 1176 array design. Consensus sequence determined for c-Raf substrates on analysis of peptide array data shows a strong selection for both hydrophobic and basic residues at the −3 position. A strong preference for leucine is seen at the −2 position. Proline and arginine are strongly preferred at the −1 position. An examination of the amino acid preferences C-terminal to the fixed phosphorylated residue reveals a bias towards aspargine compared to other residues at the +1 position. Also, acyclic and hydrophobic amino acids are seen at the +1 position with no preference for any particular group of amino acid at the +2 position. The +3 position shows a strong preference for basic residues.

Mentions: The capacity of c-Raf for in vitro phosphorylation studies was examined by incubating it with MEK (Mitogen Activated Protein Kinase Kinase), a well established substrate. As evident from figure 2A, our c-Raf preparation was highly active on MEK and we decided to test its ability to phosphorylate peptides immobilized in an array format containing 1176 phosphobase-derived peptides (see materials and methods). A one hour incubation with c-Raf resulted in extensive peptide phosphorylation, with different peptides incorporating wildly different amounts of 33P, demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation (figure 2B). Subsequent analysis was performed to see whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation.


Comparison of peptide array substrate phosphorylation of c-Raf and mitogen activated protein kinase kinase kinase 8.

Parikh K, Diks SH, Tuynman JH, Verhaar A, Löwenberg M, Hommes DW, Joore J, Pandey A, Peppelenbosch MP - PLoS ONE (2009)

Analysis of phosphorylation of 1176 peptide array by c-Raf.A. In vitro phosphorylation of MEK by c-Raf. The capacity of purified c-Raf for in vitro phosphorylation studies was examined by incubating purified c-Raf with MEK and detected using MEKSer218/222/MEK2Ser222/226 antibodies. B. c-Raf phosphorylation of 1176 peptide array. Phosphorylation of the 1176 peptide array, spotted in duplicate, on incubation with c-Raf and 33P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation. Further analysis was carried out to determine whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation. C. Consensus sequence of c-Raf substrates using 1176 array design. Consensus sequence determined for c-Raf substrates on analysis of peptide array data shows a strong selection for both hydrophobic and basic residues at the −3 position. A strong preference for leucine is seen at the −2 position. Proline and arginine are strongly preferred at the −1 position. An examination of the amino acid preferences C-terminal to the fixed phosphorylated residue reveals a bias towards aspargine compared to other residues at the +1 position. Also, acyclic and hydrophobic amino acids are seen at the +1 position with no preference for any particular group of amino acid at the +2 position. The +3 position shows a strong preference for basic residues.
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Related In: Results  -  Collection

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pone-0006440-g002: Analysis of phosphorylation of 1176 peptide array by c-Raf.A. In vitro phosphorylation of MEK by c-Raf. The capacity of purified c-Raf for in vitro phosphorylation studies was examined by incubating purified c-Raf with MEK and detected using MEKSer218/222/MEK2Ser222/226 antibodies. B. c-Raf phosphorylation of 1176 peptide array. Phosphorylation of the 1176 peptide array, spotted in duplicate, on incubation with c-Raf and 33P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation. Further analysis was carried out to determine whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation. C. Consensus sequence of c-Raf substrates using 1176 array design. Consensus sequence determined for c-Raf substrates on analysis of peptide array data shows a strong selection for both hydrophobic and basic residues at the −3 position. A strong preference for leucine is seen at the −2 position. Proline and arginine are strongly preferred at the −1 position. An examination of the amino acid preferences C-terminal to the fixed phosphorylated residue reveals a bias towards aspargine compared to other residues at the +1 position. Also, acyclic and hydrophobic amino acids are seen at the +1 position with no preference for any particular group of amino acid at the +2 position. The +3 position shows a strong preference for basic residues.
Mentions: The capacity of c-Raf for in vitro phosphorylation studies was examined by incubating it with MEK (Mitogen Activated Protein Kinase Kinase), a well established substrate. As evident from figure 2A, our c-Raf preparation was highly active on MEK and we decided to test its ability to phosphorylate peptides immobilized in an array format containing 1176 phosphobase-derived peptides (see materials and methods). A one hour incubation with c-Raf resulted in extensive peptide phosphorylation, with different peptides incorporating wildly different amounts of 33P, demonstrating that peptide sequences confer specificity to c-Raf-dependent phosphorylation (figure 2B). Subsequent analysis was performed to see whether the primary sequence of the peptides employed revealed information as to the amino acid preferences of this enzyme for substrate phosphorylation.

Bottom Line: Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology.The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes.Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Section Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. k.parikh@med.umcg.nl

ABSTRACT
Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

Show MeSH