Proteomic Analysis of Calcium- and Phosphorylation-dependent Calmodulin Complexes in Mammalian Cells

Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed “Dynamic Phosphoprotein Complex Trapping”, “DPPC Trapping” for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulinbinding proteins are then trapped in vitro in a Ca-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitinprotein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP3R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP3R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca-dependent CaM binding. However, pSer/Thr of IP3R1 did not substantially affect its Ca-indepedent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B, plays a critical role in modulating the