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

Nagib Ahsan, Yadong Huang, Alejandro Tovar-Mendez, Kirby N. Swatek, Jingfen Zhang, Ján A. Miernyk, Dong Xu, Jay J. Thelen

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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 multiple-sites by three distinct PK, casein kinase1-like 10, AME3, 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.

Original languageEnglish (US)
Pages (from-to)937-948
Number of pages12
JournalJournal of Proteome Research
Volume12
Issue number2
DOIs
StatePublished - Feb 1 2013

Keywords

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

Fingerprint Dive into the research topics of 'A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology'. Together they form a unique fingerprint.

Cite this