Comparison of peptide array substrate phosphorylation of c-Raf and mitogen activated protein kinase kinase kinase 8.
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.
Parikh
Kaushal
K
Department of Cell Biology, Section Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. k.parikh@med.umcg.nl
Diks
Sander H
SH
Tuynman
Jurriaan H B
JH
Verhaar
Auke
A
Löwenberg
Mark
M
Hommes
Daan W
DW
Joore
Jos
J
Pandey
Akhilesh
A
Peppelenbosch
Maikel P
MP
D000595:Amino Acid Sequence; D000818:Animals; D020930:MAP Kinase Kinase Kinases; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D020930:MAP Kinase Kinase Kinases; C489843:Map3k8 protein, mouse