Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation.
This chapter describes the development and implementation of three independent imaging assays for the major mitogen-activated protein kinase (MAPK) signaling modules: p38, JNK, and ERK. There are more than 500 protein kinases encoded in the human genome that share an ATP-binding site and catalytic domain conserved in both sequence and structure. The majority of kinase inhibitors have been found to be competitive with ATP, raising concerns regarding kinase selectivity and potency in an environment of millimolar intracellular concentrations of ATP, as well as the potential for off-target effects via the many other cellular proteins that bind and/or utilize ATP. The apparent redundancy of the kinase isoforms and functions in the MAPK signaling modules present additional challenges for kinase inhibitor selectivity and potency. Imaging assays provide a method to address many of these concerns. Cellular imaging approaches facilitate analysis of the targets expressed in the context of their endogenous substrates and scaffolding proteins and in a complex environment for which subcellular localization, cross talk between pathways, phosphatase regulatory control, and intracellular ATP concentrations are relevant to the functions of the kinase. The assays described herein provide a strategy to profile kinase inhibitors for MAPK pathway selectivity while simultaneously providing information on cell morphology or toxicity. Results suggest that the MAPK pathways are indeed susceptible to nonselective kinase inhibitors such as staurosporin and inhibitors that inhibit upstream MAPK Kinase Kinases (MKKKs) and MAPK Kinases (MKKs) in the MAPK signaling pathway, especially those involved in cross talk between the pathways. However, selective MAPK inhibitors were identified that exhibited pathway selectivity as evidenced by significantly lower IC(50) values for their respective p38, JNK, or ERK signaling pathway assays.
Nickischer
Debra
D
Sphinx RTP Laboratories, Eli Lilly and Company, Research Triangle Park, NC, USA.
Laethem
Carmen
C
Trask
Oscar J
OJ
Williams
Rhonda Gates
RG
Kandasamy
Ramani
R
Johnston
Patricia A
PA
Johnston
Paul A
PA
D001671:Biochemistry / Q000379:methods*; D020134:Catalytic Domain; D002454:Cell Differentiation; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System; D020033:Protein Isoforms; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099190:MAP-Kinase-Activated Kinase 2
D048049:Extracellular Signal-Regulated MAP Kinases; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016755:Proto-Oncogene Proteins c-jun; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2