TY - JOUR
T1 - Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry
AU - Ahsan, Nagib
AU - Kataya, Amr R. A.
AU - Rao, R. Shyama Prasad
AU - Swatek, Kirby N.
AU - Wilson, Rashaun S.
AU - Meyer, Louis J.
AU - Tovar-Mendez, Alejandro
AU - Stevenson, Severin
AU - Maszkowska, Justyna
AU - Dobrowolska, Grazyna
AU - Yao, Qiuming
AU - Xu, Dong
AU - Thelen, Jay J.
N1 - Publisher Copyright:
© 2024 by the authors.
PY - 2024/6/1
Y1 - 2024/6/1
N2 - Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.
AB - Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.
KW - kinase client assay
KW - kinase substrates
KW - mass spectrometry
KW - phosphorylation
KW - signaling network
UR - http://www.scopus.com/inward/record.url?scp=85195806148&partnerID=8YFLogxK
U2 - 10.3390/plants13111481
DO - 10.3390/plants13111481
M3 - Article
C2 - 38891291
SN - 2223-7747
VL - 13
JO - Plants
JF - Plants
IS - 11
M1 - 1481
ER -