Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

The mitogen-activated protein kinase (MAPK) signaling cascade involves a hierarchy of kinases that activate one another through consecutive phosphorylation events in response to extracellular or intracellular signals [15] . Standard methods have only been able to establish a few combinatorial connections from upstream MKK-activating kinases (MKKKs) to downstream MPK-activating kinases (MKKs), MAPKs and their cytoplasmic and nuclear substrates [26, 27] . Constructing this complicated interconnected network necessitates a systematic unbiased high-throughput approach to avoid confounding issues of redundancy and functional pleiotropy [15] . Akin to the protein microarray based kinase assays developed by Ptacek et al. [20] , Popescu et al. employed high-density protein microarrays to identify novel MPK substrates. The authors first determined which Arabidopsis thaliana MKKs preferentially activate 10 different MPKs in vivo and used the activated MPKs to probe Arabidopsis protein microarrays containing 2158 unique proteins to reveal their phosphorylation substrates [15] . The initial screen identified 570 nonredundant MPK phosphorylation substrates with an average of 128 targets per activated MPK. With this data the authors were able to reconstruct a complex signaling cascade involving nine MKKs, 10 MPKs and 570 substrates [15] . Moreover, the resulting nodes and edges highlighted the specificity conserved within these interactions: 290 (51%) of MPK phosphorylation targets were hit by only one MPK and only 94 (16%) were phosphorylated by two or more MPKs [15] . Gene ontology (GO) analysis of effector substrates showed enrichment in TFs involved in the regulation of development, defense and stress responses [15] . The network that emerged from this study suggests the MAPK signaling cascade regulates transcription through combinatorial enzyme specificity and discrete phosphorylation events.