Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca 2+ release and apoptosis (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 2008
The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP 3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol P i incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through G s-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through G q-coupled muscarinic receptors) or other Ca 2؉ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP 3-induced Ca 2؉ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP 3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both G sand G q-coupled cell surface receptors to effect InsP 3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca 2؉ mobilization.
Cell Signalling: IP3 Receptors Channel Calcium into Cell Death
Current Biology, 2004
There is substantial evidence that Ca 2+ fluxes occur during most forms of apoptosis, and that inhibiting such fluxes protects cells from death. IP 3 receptorsligand-gated channels that release Ca 2+ from intracellular stores -are emerging as key sites for regulation by pro-and anti-apoptotic factors.
Inositol Hexakisphosphate Kinase-2, a Physiologic Mediator of Cell Death
Journal of Biological Chemistry, 2005
Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by a family of three inositol hexakisphosphate kinases (InsP6K). In this study we establish one of the InsP6Ks, InsP6K2, as a physiologic mediator of cell death. Overexpression of wild-type InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas transfection with a dominant negative InsP6K2 decreases cell death. During cell death, InsP6 kinase activity is enhanced, and intracellular InsP7 level is augmented. Deletion of InsP6K2 but not the other forms of InsP6K diminishes cell death, suggesting that InsP6K2 is the major InsP6 kinase involved in cell death. Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process.
Biochemical and Biophysical Research Communications, 2008
The proto-oncogene AKT is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca 2+ ) transfer from the Endoplasmic Reticulum (ER) to the mitochondria in several models of apoptosis. AKT is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.
Proceedings of the National Academy of Sciences of the United States of America, 2013
K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho-K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL-dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP3 regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurviv...
Proceedings of the National Academy of Sciences, 2004
Proapoptotic BCL-2 family members BAX and BAK are required for the initiation of mitochondrial dysfunction during apoptosis and for maintaining the endoplasmic reticulum (ER) Ca 2+ stores necessary for Ca 2+ -dependent cell death. Conversely, antiapoptotic BCL-2 has been shown to decrease Ca 2+ concentration in the ER. We found that Bax -/- Bak -/- double-knockout (DKO) cells have reduced resting ER Ca 2+ levels because of increased Ca 2+ leak and an increase in the Ca 2+ -permeable, hyperphosphorylated state of the inositol trisphosphate receptor type 1 (IP3R-1). The ER Ca 2+ defect of DKO cells is rescued by RNA interference reduction of IP3R-1, supporting the argument that this channel regulates the increased Ca 2+ leak in these cells. BCL-2 and IP3R-1 physically interact at the ER, and their binding is increased in the absence of BAX and BAK. Moreover, knocking down BCL-2 decreases IP3R-1 phosphorylation and ER Ca 2+ leak rate in the DKO cells. These findings support a model in ...
Biochemical Journal, 2005
IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.
Journal of Biological Chemistry, 1999
The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R), an IP 3 -gated Ca 2؉ channel located on intracellular Ca 2؉ stores, modulates intracellular Ca 2؉ signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP 3 R type 1 (IP 3 R1) was found to be cleaved. IP 3 R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH 2 DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP 3 R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP 3 R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888 DEVD* 1892 R (mouse IP 3 R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP 3 R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP 3 R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-␣-or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP 3 R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP 3 R1 degradation upon apoptotic stimuli. Therefore IP 3 R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP 3 R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP 3 R1 was digested, indicating that caspase-3 inactivates IP 3 R1 channel functions.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is a universal intracellular Ca 2+ -release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca 2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca 2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP 3 R or with associated proteins and so provide a large potential for fine tuning the Ca 2+ -release activity and for creating efficient Ca 2+ signals in subcellular microdomains.