Identification of Novel Substrates of MAP Kinase Cascades Using Bioengineered Kinases that Uniquely Utilize Analogs of A
The Mitogen-Activated Protein Kinase (MAPK) family of signaling molecules regulates a number of cellular processes through the direct phosphorylation and regulation of a plethora of cellular proteins. Identifying the direct substrates of the MAPK pathway
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oduction The MAP kinase (MAPK) intracellular signaling cascades are activated by a number of extracellular and intracellular stimuli, including growth factors, cytokines, cell stress, cell adhesion, chemotherapeutic drugs, reactive oxygen species, and irradiation. MAPK activation has been linked to a number of cellular processes, including proliferation, migration, apoptosis, and Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_10, © Springer Science+Business Media, LLC 2010
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Zheng, Al-Ayoubi, and Eblen
ifferentiation. In order to understand how MAPKs have so d many effects on cellular physiology, one must first identify the specific targets of MAPK pathways and how the temporal phosphorylation of the substrates that they modify controls the diverse array of biological responses that occur in response to MAPK activation. While many MAPK substrates have been identified, identification of novel substrates is a very active area of investigation. In this chapter, we focus on the identification of novel substrates of MAPK signaling by the use of kinases mutated to allow them to utilize analogs of ATP to phosphorylate their direct substrates, a technique that was originally developed by Kevan Shokat and Kavita Shah for use with Src tyrosine kinase (1). We have utilized this technique to modify the MAPKs ERK2 (extracellular regulated kinase 2) (2) and p38a, as well as MAPK kinase MEK1/ MKK1. This technique has been utilized by others on Raf-1 (3), Jun N-terminal kinase 1 (JNK1) (4), and p38a (5). The technique for identifying novel kinase substrates relies on the observation that protein kinases have structurally similar ATPbinding domains, with most containing one or more amino acids with a large side chain at a key conserved position(s) that helps regulate the size of the domain around the N6 position of ATP (Fig. 1a). This amino acid, termed the “gatekeeper” residue (1), comes into close contact with the N6 position of bound ATP. Mutation of this residue to a smaller amino acid, such as glycine or alanine, creates extra space in the ATP binding site around the
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ERK2 p38α MEK1 JNK Raf1
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ERK2 p38α MEK1 JNK Raf1
QMKDVYIVQDLMET-108 EFNDVYLVTHLMGA-111 SDGEISICMEHMDG-148 EFQDVYIVMELMDA-113--TLKILDFGL-172 TKDNLAIVTQWCEG-426--NNIFLHEGL-480 Q103G T106G M143G M108G/L168A T421A/F475L
Fig. 1. Alignment of MAPK family gatekeeper mutations. (a) ATP, with an arrow pointing to the N6 position. (b) Alignment of MAPK family proteins, with the gatekeeper residues in bold italicized. (c) ATP-binding domain mutations in MAPK family proteins.
Identification of Novel Substrates of MAP Kinase Cascades
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N6-position of ATP (1). The increased size of the ATP binding site allows the kinase to still utilize ATP in most cases and also allows the mutant kinase to utilize ATP analogs that have a bulky side group chemically synthesized onto the N6 position. Depending on the N6-ATP analog, the wild-type kinase and other cellular protein
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