Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs.
The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified by differential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade.
Eblen
Scott T
ST
Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. mjw@virginia.edu
Kumar
N Vinay
NV
Shah
Kavita
K
Henderson
Michelle J
MJ
Watts
Colin K W
CK
Shokat
Kevan M
KM
Weber
Michael J
MJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D004268:DNA-Binding Proteins; D006801:Humans; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D028861:Nuclear Pore Complex Proteins; D010453:Peptide Synthases / Q000378:metabolism; D010766:Phosphorylation; D015202:Protein Engineering; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors; D014162:Transfection; D044767:Ubiquitin-Protein Ligases; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; D028861:Nuclear Pore Complex Proteins; D011518:Proto-Oncogene Proteins; C073597:TPR protein, human; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D000255:Adenosine Triphosphate; C117617:UBR5 protein, human; D044767:Ubiquitin-Protein Ligases; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D019950:Mitogen-Activated Protein Kinase 1; D010453:Peptide Synthases