Cdc2/cyclin B1 interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions (original) (raw)
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Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by Cdc2
Journal of Cellular Biochemistry, 2003
Calcium (Ca 2þ ) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca 2þ -release channels found in many cell types. Although IP3R-mediated Ca 2þ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca 2þ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin-dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser 421 and Thr 799 and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.
Biochemical and Biophysical Research Communications, 2006
Calcium (Ca 2þ) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca 2þ-release channels found in many cell types. Although IP3R-mediated Ca 2þ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca 2þ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin-dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser 421 and Thr 799 and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.
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 Calcium, 2006
The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R1) function as a suppressor/coupling domain. In this study we used IP 3 R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP 3 binding and Ca 2+ -release activity. Although the N-terminal 1-225 aa of IP 3 R3 had the same role as in IP 3 R1, the suppression of IP 3 binding for IP 3 R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP 3 -induced activation (IICR). Deletion of 11 aa in IP 3 R1 ([ 76-86]-IP 3 R1) or replacing aa 76-86 of the IP 3 R1 in the suppressor/coupling domain by 13 aa of IP 3 R3 ([75-87 T3]-IP 3 R1) also resulted in increased IP 3 binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [ 76-86]-IP 3 R1 and of [75-87 T3]-IP 3 R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca 2+ content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca 2+ -release activity of IP 3 R1 to IP 3 influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR. 3 R1 can be divided in two parts. The minimal sequence needed for the binding of IP 3 is located between amino acids 226-578 (mouse sequence) and is called IP 3 -binding core . Its boomerang-like crystal structure in complex with its ligand consists of an N-terminal -trefoil fold and a C-terminal ␣-helical subdomain containing an "armadillo repeat"-like fold . The affinity of this IP 3 -binding core for IP 3 is more than 10-fold higher than that of the full IP 3 -binding domain where the 1-225 Nterminal residues are included . In addition, these 1-225 residues were also found to be critical for IP 3 R functioning . This aa 1-225 domain was therefore referred to as a suppressor/coupling domain. The crystal structure of 0143-4160/$ -see front matter
Proceedings of the National Academy of Sciences, 2007
Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca 2+ homeostasis. Antiapoptotic Bcl-x L binds to the inositol trisphosphate receptor (InsP 3 R) Ca 2+ release channel to enhance Ca 2+ - and InsP 3 -dependent regulation of channel gating, resulting in reduced ER [Ca 2+ ], increased oscillations of cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ), and apoptosis resistance. However, it is controversial which InsP 3 R isoforms mediate these effects and whether reduced ER [Ca 2+ ] or enhanced [Ca 2+ ] i signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP 3 R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP 3 R. In contrast, coexpression of each isoform with Bcl-x L conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER...
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.
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.