Identification of Protein Substrates of Ca2+/Calmodulin-Dependent Protein Kinase II in the Postsynaptic Density by Protein Sequencing and Mass Spectrometry

2002 ◽  
Vol 290 (3) ◽  
pp. 948-954 ◽  
Author(s):  
Yoshiyuki Yoshimura ◽  
Takashi Shinkawa ◽  
Masato Taoka ◽  
Kana Kobayashi ◽  
Toshiaki Isobe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Postsynaptic Density ◽  
Protein Sequencing ◽  
Dependent Protein Kinase ◽  
Protein Substrates ◽  
Protein Kinase Ii ◽  
Dependent Protein

scholarly journals The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta

FEBS Letters ◽  
2008 ◽  
Vol 582 (13) ◽  
pp. 1795-1801 ◽  
Author(s):  
Takashi Masaoka ◽  
Mayuko Nishi ◽  
Akihide Ryo ◽  
Yaeta Endo ◽  
Tatsuya Sawasaki
Keyword(s):  
Protein Kinase ◽  
Germ Cell ◽  
Wheat Germ ◽  
Dependent Protein Kinase ◽  
Protein Substrates ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

scholarly journals Wnt5a Induces Association of ROR1 with Ca2+/Calmodulin-Dependent Protein Kinase II and ROR1-Dependent Calcium Influx in Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1846-1846
Author(s):  
Liguang Chen ◽  
Yun Chen ◽  
Jian Yu ◽  
Ling Zhang ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Conflicts Of Interest ◽  
Calcium Influx ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could enhance CLL-cell proliferation (Yu J, et al, JCI 126:585, 2016; Yu J, et al, Leukemia 31:2608, 2017; Hasan MK, et al, Blood 132:170, 2018). We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine-specific protein kinase. Recent studies demonstrated that high levels of different isoforms of CaMK, especially CaMKII, were expressed in several cancers. CaMKII phosphorylates nearly 40 different proteins, including enzymes, ion channels, kinases, and transcription factors and can play a critical role in the regulation of proliferation and survival of various cancer cells (Wang YY, et al, Oncotarget 20:11725, 2015; Chi M, et al, Sci Rep 6:33132, 2016; Hoffman A, et al, Cell Signal 26:748, 2014). We validated our mass spectrometry finings in co-immunoprecipitation studies and immunoblot analyses. These studies showed that Wnt5a could induce the specific phosphorylation and recruitment of CaMKII to ROR1. Moreover, these studies demonstrated that Wnt5a-induced recruitment of CaMKII to ROR1 could be blocked by cirmtuzumab, a first-in-class humanized anti-ROR1 mAb undergoing clinical testing in patients with CLL (Choi MY, et al, Cell Stem Cell 22:951, 2018). CaMKs serve as a ubiquitous intracellular receptor for Ca2+ and their activity are regulated through Ca2+ signaling. Therefore, we examined calcium influx in CLL cells by flow cytometry. Treatment of CLL cells cultured overnight in serum-free medium induced calcium influx within seconds. Moreover, the relative capacity of Wnt5a to induce calcium influx was associated with the relative expression level of ROR1 on CLL cells of different patients. Furthermore, Wnt5a could enhance calcium influx induced by treatment with anti-µ in CLL cells that expressed ROR1, but not in CLL cells that lacked expression of ROR1. Knockdown of ROR1 using ROR1 siRNA or treatment with cirmtuzumab inhibited the capacity of Wnt5a to enhance calcium influx induced by treatment with anti-µ. Collectively, these data demonstrate that Wnt5a-induced ROR1-signaling in CLL can influence BCR-signaling and activation of the Ca2+/calmodulin-dependent protein kinase II signaling pathway. Disclosures No relevant conflicts of interest to declare.

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