Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by Cdc2

Calcium (Ca2+) 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 Ca2+-release channels found in many cell types. Although IP3R-mediated Ca2+ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca2+ 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.     

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non- muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation- contraction coupling in the heart.     

HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis

Caspase-3 (CASP3) plays a key role in apoptosis. In this study, HAX-1 was identified as a new substrate of CASP3 during apoptosis. HAX-1 was cleaved by CASP3 during etoposide-(ETO) induced apoptosis, and this event was inhibited by a CASP3-specific inhibitor. The cleavage site of HAX-1, at Asp(127), was located using N-terminal amino acid sequencing of in vitro cleavage products of recombinant HAX-1. Overexpression of HAX-1 inhibited ETO-induced apoptotic cell death. It also inhibited CASP3 activity. Together, these results suggest that HAX-1, a substrate of CASP3, inhibits the apoptotic process by inhibiting CASP3 activity.     

Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs

To initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca²⁺]_i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP₃R1), the channel implicated, undergoes modifications that enhance its function. We found that IP₃R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca²⁺]_i responses cease. We also reported that maturation without ERK activity diminishes IP₃R1 MPM-2 reactivity and [Ca²⁺]_i responses. Here, we show that IP₃R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP₃R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP₃R1 cortical re-distribution. We propose that IP₃R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca²⁺]_i signals during meiosis/mitosis and cytokinesis.     

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.     

Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis

We have previously shown that Xenopus rabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J. 16, 6182-6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5.     

Degradation of the type I inositol 1,4,5-trisphosphate receptor by caspase-3 in SH-SY5Y neuroblastoma cells undergoing apoptosis

The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.     

Protein disulfide isomerase is cleaved by caspase-3 and -7 during apoptosis

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.     

Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP(3)R) results in a more pronounced Ca(2+)release in the presence of inositol 1,4,5-trisphosphate (IP(3)). Two recently published studies demonstrated a different ATP sensitivity of IP(3)-induced Ca(2+)release in cell types expressing different IP(3)R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to ATP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP(3)receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem 1998;273: 8983-8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca(2+)signals by differential expression of IP(3)receptor subtypes. EMBO J 1999;18: 1303-1308). In order to investigate the difference in ATP sensitivity between IP(3)R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or IP(3)R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[alpha-(32)P]ATP. ATP labeling of the IP(3)R was measured after immunoprecipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-(32)P]ATP to the recombinant IP(3)R1 and IP(3)R3 with an IC(50)of 1.6 microM and 177 microM, respectively. MgATP was as effective as ATP in displacing 8-azido[alpha-(32)P]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimulating IP(3)-induced Ca(2+)release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP(3)-binding domains, since ATP inhibited IP(3)binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3)R3 with an IC(50)of 353 microM and 4.0 mM, respectively. The ATP-binding sites of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, while IP(3)R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP.     

Inositol 1,4,5-trisphosphate (IP3) responsiveness is regulated in a meiotic cell cycle dependent manner: implications for fertilization induced calcium signaling

Fertilisation of mammalian eggs is known to trigger a series of transient rises in cytosolic calcium, known as calcium oscillations, the initiation and duration of which are crucial for meiotic exit and subsequent entry into embryogenesis. It is not known how these calcium oscillations are terminated when the zygote exits meiosis; it is thought that responsiveness to inositol 1,4,5-trisphosphate (IP3) is involved, since the oscillations are known to be mediated by an IP3 dependent mechanism. Here we report that IP3 responsiveness is maintained throughout meiotic maturation and falls very rapidly after meiotic exit. We also show that inhibition of the major cell cycle kinase, CDK1, has no effect on responsiveness, but that prolonging CDK1 activity prevents the decline in responsiveness normally seen at meiotic exit. We conclude that CDK1 plays a role, but is not the only factor involved in controlling IP3 responsiveness during the meiotic cell cycle.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Inositol 1,4,5-trisphosphate receptor movement is restricted by addition of elevated levels of O-linked sugar

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a versatile, ubiquitous intracellular calcium channel. Traditionally, visualizing the InsP3R in live cells involves attaching a fluorescent marker to either terminal of the protein, but the termini themselves contain binding sites for accessory molecules and proteins. Using random transposition, constructs have been developed that express the type I InsP3R with green fluorescent protein (GFP) inserted at various points within its sequence. We have used two of these constructs, one in the ligand-binding domain, and another in the regulatory domain, to investigate InsP3R dynamics within the endoplasmic reticulum. We present evidence that endogenous calcium signaling is maintained when these constructs are expressed. In addition, by measuring the fluorescent recovery after photobleaching of a subcellular region, we demonstrate that treatment with 8mM N-acetylglucosamine (GlcNAc), known to lead to increased O-linked GlcNAcylation of proteins, leads to a reduction in the ability of the InsP3R to travel laterally within the endoplasmic reticulum. Each construct serves as the control for the other one, suggesting that this decrease in mobility is not specific to the insertion site of GFP within the InsP3R. These constructs represent a new tool that will allow us to follow receptor turnover and translocation events.     

The inositol 1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type 2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.