Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Pasan Fernando,Wen Deng,Beata Pekalska,Yves Derepentigny,Rashmi Kothary,John F Kelly,Lynn A Megeney

doi:10.1074/mcp.m400200-mcp200

Pasan Fernando, Wen Deng + Show 5 more

Open Access

https://doi.org/10.1074/mcp.m400200-mcp200

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Abstract

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

Highlights

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly
This pathway contains a phosphorylation sequence initiated by a mitogen-activated protein kinase (MAPK) kinase kinase (MKKK), which activates a MAPK kinase (MKK), which in turn activates a MAPK (p38), with the MAPK serving as the effector enzyme to stimulate or repress the activity of corresponding protein substrates by targeted phosphorylation [7]
To begin to address the depth of the MKK6/p38 signaling cascade, we chose to utilize the post-natal heart as a modeling environment

Summary

Introduction

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. Using this assay with gel-embedded substrates, we identified a broad range of kinase activities in lysates derived from the hearts of transgenic mice expressing activated MKK6. These results provide a proof of concept that KS/MS (KSE/MS and KSS/MS) is applicable for large scale identification and analyses of both protein kinase activities and their effective substrate range.

Results

Conclusion