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Title: A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology

item AHSAN, NAGIB - University Of Missouri
item HUANG, YADONG - University Of Missouri
item TOVAR-MENDEZ, ALEJANDRO - University Of Missouri
item SWATEK, KIRBY - University Of Missouri
item ZHANG, JINGFEN - University Of Missouri
item Miernyk, Jan
item XU, DONG - University Of Missouri
item THELEN, JAY - University Of Missouri

Submitted to: Journal of Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2013
Publication Date: 6/1/2013
Citation: Ahsan, N., Huang, Y., Tovar-Mendez, A., Swatek, K.N., Zhang, J., Miernyk, J.A., Xu, D., Thelen, J. 2013. A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology. Journal of Proteomics. 12(2):937-948.

Interpretive Summary: Information transfer in living organisms is often referred to as signaling. Signaling is a network phenomenon by which information is generated, transferred, decoded, and acted upon. Signaling networks can be very complex, having hundreds of members. Because a signal is increased when transferred, this process can be very efficient. The huge complexity of signaling networks has made it impossible to duplicate it in the lab. Herein we describe a new method to experimentally approximate the complexity of signaling. Relatively small synthetic strings of amino acids are used in place of the large, naturally-occurring network members. The transfer of phosphate is measured and then quantified using a specialized instrument. Using this method we were able to approximate in the lab the biological interactions that take place inside plant cells. This information will be useful to scientists in their efforts to improve agricultural crop production and improve both protein and oil quality through both classical breeding and application of biotechnology strategies.

Technical Abstract: While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made towards identifying their individual client proteins. Herein we describe use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. Based on the results from in vivo phospho-proteomic analyses of developing A. thaliana and Brassica napus seeds, a synthetic peptide library was generated comprising 377 sequences. This peptide library was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance, protein phosphatase inhibitor-2 (AtPPI-2), was phosphorylated at multiple-sites by three distinct PK. To confirm this result, full-length recombinant AtPPI-2 was phosphorylated in vitro with casein kinase1-like 10, AME3, or a Ser PK-like protein. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 has the potential to induce conformational changes that could modulated TOPP binding and/or activity. We propose that multiple signaling pathways converge at AtPPI-2, and that AtPPI-2 is phosphorylated at either distinct uni- or combined multi-sites in response to different stimuli. Overall, our data validates the utility of the KiC assay strategy in both PK-client discovery screening and in contributing to our understanding of signaling network topology.