A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

作者：Ahsan Nagib; Huang Yadong; Tovar Mendez Alejandro; Swatek Kirby N; Zhang Jingfen; Miernyk Jan A; Xu Dong; Thelen Jay J^*

来源：Journal of Proteome Research, 2013, 12(2): 937-948.

DOI：10.1021/pr3009995

• mass spectrometry
• kinase
• phosphorylation
• protein-protein interaction
• phosphatase inhibitor
• signaling network

While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed 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 multiplesites by three distinct PK, casein kinase1-like 10, ANLE3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK 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 induces conformational changes that modulate TOPP binding.

• 出版日期2013-2