title: Proteomic and biochemical studies of calcium- and phosphorylation-dependent calmodulin complexes in Mammalian cells.
abstract: 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.
authors: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 84-171, Berkeley, California 94720, USA.
authors: Deok-Jin
authors: DJ
authors: Jang
authors: Mingquan
authors: M
authors: Guo
authors: Daojing
authors: D
authors: Wang
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011720:Pyrazoles / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D002147:Calmodulin; D004791:Enzyme Inhibitors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011506:Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D025801:Ubiquitin; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases; D019311:Staurosporine; D002118:Calcium