Computational prediction and experimental verification of new MAP kinase docking sites and substrates including Gli transcription factors.
In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new 'D-site' class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.
Whisenant
Thomas C
TC
Department of Developmental and Cell Biology, University of California, Irvine, California, United States of America.
Ho
David T
DT
Benz
Ryan W
RW
Rogers
Jeffrey S
JS
Kaake
Robyn M
RM
Gordon
Elizabeth A
EA
Huang
Lan
L
Baldi
Pierre
P
Bardwell
Lee
L
D000465:Algorithms; D001185:Artificial Intelligence; D001665:Binding Sites; D015894:Genome, Human; D006801:Humans; D051188:Knowledge Bases; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry; D000071676:Zinc Finger Protein GLI1; D000074290:Zinc Finger Protein Gli3
C400551:GLI1 protein, human; C065683:GLI3 protein, human; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D000074290:Zinc Finger Protein Gli3; D020928:Mitogen-Activated Protein Kinases