Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor.
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 approximately 8-fold over wild type and approximately 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 approximately 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 alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-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.
Wagner
Larry E
LE
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, USA.
Li
Wen-Hong
WH
Joseph
Suresh K
SK
Yule
David I
DI
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D002118:Calcium