Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (44), p.46242-46252
• Main Authors: Wagner, 2nd, Larry E, Li, Wen-Hong, Joseph, Suresh K, Yule, David I
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium Channels - physiology; Calcium Signaling; Cyclic AMP-Dependent Protein Kinases - metabolism; Cyclic GMP-Dependent Protein Kinases - metabolism; Humans; Inositol 1,4,5-Trisphosphate Receptors; Mutation; Phosphorylation; Rats; Receptors, Cytoplasmic and Nuclear - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M405849200

Regulation of Ca 2+ release through inositol 1,4,5-trisphosphate receptors (InsP 3 R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca 2+ signals. In this study, regulation of Ca 2+ release by phosphorylation of type 1 InsP 3 R (InsP 3 R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP 3 R-1 splice variants. Ca 2+ release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP 3 R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP 3 R-1, InsP 3 -induced Ca 2+ release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca 2+ release through the S2- S1589E/S1755E InsP 3 R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP 3 R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca 2+ release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca 2+ release. The sensitivity of Ca 2+ release in cells expressing S2+ S1755E InsP 3 R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP 3 R-1. In contrast, mutation of S2+ S1589E InsP 3 R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP 3 R-1 resulted in robust Ca 2+ oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP 3 R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca 2+ oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP 3 R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.