Abstract
Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.
Highlights
Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells
Activation or inhibition can be achieved by kinase phosphorylation [e.g., mitogen-activated protein kinases (MAPKs), glycogen synthase kinase-3 (GSK3)] or allosteric protein-protein interactions [cyclin-dependent kinases (CDKs), protein kinase A (PKA)]
Net cellular activity is altered by protein stabilization [nuclear factor-κB-inducing kinase (NIK)] or transcriptional upregulation [proviral integrations of moloney (PIM) kinases]
Summary
Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. A kinase-substrate pair recognized for its specificity is the Thr-Tyr bisphosphorylation of extracellular-regulated kinase (ERK) by MAPK-ERK kinase (MEK)[10]. Full activation of some kinases, such as Akt and c-jun N-terminal kinase (JNK), requires separate phosphorylation events that are catalyzed [18,19] or preferentially[20] by different upstream kinases. Mass spectrometry has quantified in vitro phosphorylation of multiple peptide substrates in whole cell extracts[26,27], but unfractionated assays cannot attribute activity to individual kinases[28]. Prior attempts with in vitro kinase biochemistry were unable to assign substrate-phosphorylating activities cleanly to specific kinases[26,27,28,37]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
