title: A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.
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.
authors.affiliation: Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Nagib
authors.initials: N
authors.lastname: Ahsan
authors.forename: Yadong
authors.initials: Y
authors.lastname: Huang
authors.forename: Alejandro
authors.initials: A
authors.lastname: Tovar-Mendez
authors.forename: Kirby N
authors.initials: KN
authors.lastname: Swatek
authors.forename: Jingfen
authors.initials: J
authors.lastname: Zhang
authors.forename: Ján A
authors.initials: JA
authors.lastname: Miernyk
authors.forename: Dong
authors.initials: D
authors.lastname: Xu
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Thelen
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D000097847:Protein Phosphatase Inhibitory Proteins
chemical_list: D029681:Arabidopsis Proteins; D019151:Peptide Library; D010750:Phosphoproteins; D011494:Protein Kinases; D054645:Protein Phosphatase 1; D011506:Proteins; D011994:Recombinant Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins