Proteomic and biochemical studies of calcium- and phosphorylation-dependent calmodulin complexes in Mammalian cells.

Protein conformational changes due to cofactor binding (e.g., metal ions, heme) and/or post-translational modifications (e.g., phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium signaling and homeostasis. Herein, we report a straightforward and systematic approach to identify potential calcium- and phosphorylation-dependent CaM complexes in a proteome-wide manner. We have identified over 120 CaM-associated proteins encompassing four different classes of CaM binding in HeLa cells, namely, calcium-dependent and phosphorylation-dependent (e.g., EDD1), calcium-dependent and phosphorylation-independent (e.g., myosin IE), calcium-independent and phosphorylation-dependent (e.g., DDX3), and calcium-independent and phosphorylation-independent (e.g., DDX5). To demonstrate the utility of our method in understanding biological pathways, we showed that in vivo phosphorylation of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) at Ser1598 significantly reduced the affinity of its Ca2+-dependent CaM binding. However, phosphorylation of IP3R1 did not substantially affect its Ca2+-independent CaM binding. These results shed new lights on the mechanism underlying the marked increase of Ca2+ release due to IP3R1 phosphorylation. We further showed that staurosporine-sensitive kinase(s) and phosphatase PP1 play a critical role in modulating the phosphorylation-dependent CaM binding of IP3R1. Our method may serve as a general strategy to identify and characterize phosphorylation-dependent protein complexes, to pinpoint the phosphorylation sites and associated kinase(s) and phosphatase(s) involved in the protein-protein interactions, and to functionally characterize these complexes in mammalian cells.