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.
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, USA.
Larry E
LE
Wagner
Wen-Hong
WH
Li
Suresh K
SK
Joseph
David I
DI
Yule
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