INTRODUCTION

Inositol 1,4,5-trisphosphate (IP3) is an important second messenger in animal cells that mediates calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol 1, 2, 3. Inositol 1,3,4,5-tetrakisphopsphate (IP4) is another messenger responsible for mediating Ca2+ entry through plasma membrane and mobilize intracellular Ca2+ by acting synergistically with IP3 4. Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) phosphorylates IP3 to IP4 1, 5. Thus, IP3K plays a key role in maintaining Ca2+ homeostasis by regulating the concentrations of IP3 and IP4.

IP3 also serves as a precursor for the synthesis of other higher inositol phosphate (IP) isomers in IP metabolism 6, 7. These water-soluble IP isomers are involved in multiple cellular events such as modulating Ras GTPase-activating protein 8, blocking tumor cell growth 9, regulating mRNA export 10 and so on. In addition, inositol 1,2,3,4,5,6-hexakisphosphate (IP6) is related to human neutrophil function 11 and plant seed germination 12, 13. Yeast and Arabidopsis IP3Ks, also referred to as inositol polyphosphate kinase (Ipk) and inositol phosphate multikinase (Ipmk), recognize IP3 as substrate and add a phosphate to position 6 on the inositol ring to generate inositol 1,4,5,6-tetrakisphosphate (I(1,4,5,6)P4) 10, 14, 15. It is further phosphorylated by yeast and Arabidopsis IP3Ks to produce inositol 1,3,4,5,6-pentakisphosphate (IP5) 10, 14, 15. Therefore, the physiological function of IP3K is not only regulating intracellular Ca2+ homeostasis, but also controlling IP metabolism (Fig. 1).

Figure 1
figure 1

IP3K function in inositol metabolism and calcium homeostasis. Panel A shows the structure of inositol 1,4,5-trisphosphate (IP3). Panel B shows IP3K's role in phosphorylating IP3 to IP4 and IP5. IP3K regulates intracellular calcium homeostasis by controlling the balance of IP3 and IP4.

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MOLECULAR CLONING OF IP3K

Inostiol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase)

The first IP3 3-kinase cDNA (RnIP33K-A) was isolated from rat brain in 1990 16, 17, 18. Afterwards, several cDNAs encoding IP3 3-kinase were consequently cloned from human (HsIP33K-A, HsIP33K-B, HsIP33K-C) and rat (RnIP33K-B, RnIP33K-C) 19, 20, 21, 22, 23. Rat RnIP33K-B is 204 amino acids longer than that of the human HsIP33K-B, but remaining similar to its human homologue with 93% identity in amino acids 21. The recently identified human HsIP33K-C shares a highly conserved catalytic domain with human isoforms A and B 22, 23. It is about 75% identical to rat RnIP33K-C 22, 23. IP3 3-kinase from chicken 24, nematode 25 and fruit fly 26 has also been identified.

There are at least three distinct IP3 3-kinase isoforms (A, B, and C). They are different in their molecular masses, Ca2+/calmodulin (Ca2+/CaM) sensitivity, intracellular distribution and tissue expression 23, 27 (Tab. 1). Mammalian IP3 3-kinases are usually activated by Ca2+/ CaM 28. Nematode IP3 3-kinase instead lacks a consensus CaM-binding site and thus is insensitive to Ca2+/CaM 25. There are evidences suggesting that the N-terminal sequence of IP3 3-kinases is involved in intracellular localization 27, 29. For example, the N-terminal 320 amino acid sequence of rat RnIP33K-B is unique and is necessary for the binding of rat RnIP33K-B to the cytosolic face of the ER membrane 30. IP3 3-kinase isoforms show tissue specificity, such as rat RnIP33K-A is specifically expressed in brain and testes, whereas rat RnIP33K-B is predominantly expressed in lung and also in thymus, heart, testes and brain 29. Such specified distribution and expression pattern of IP3 3-kinases may contribute to their various physiological functions. However, all these IP3 3-kinases seem to have strict biochemical activity in phosphorylating IP3 to IP4 25, 26, 27.

Table 1 Biochemical and molecular characteristics of IP3K isoforms from human, rat and Arabidopsis.
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Inositol phosphate multikinase (Ipmk)/inositol polyphosphate kinase (Ipk)

Inositol phosphate multikinase (Ipmk) is widely distributed in the kingdoms of animal, plant and yeast 31. The first identified Ipmk cDNA (also called Ipk2) was from yeast 10, 14. Yeast Ipk2/Ipmk/IP3K is a dual-specificity IP3/IP4 6/3-kinase and identical to Arg82/ArgRIII which is an indispensable component of ArgR-Mcm1 transcriptional complex 10. The ArgR-Mcm1 complex functions in transcriptional control of genes involved in arginine metabolism 32, 33. However, the inositol phosphorylation activity Arg82 is not required for the transcriptional regulation 34. We previously reported the molecular cloning and characterization of a plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis 15. The amino acid sequence of AtIpk2β shares 73% identity and 84% similarity to that of a second Arabidopsis IP3K, AtIpk2α. 15, 35. Similar to yeast IP3K, Arabidopsis IP3K is also a dual-specificity 6/3-kinase 15, 35. York and his colleagues reported that Arabidopsis IP3K has a novel 5-kinase activity to phosphate I(1,3,4,6)P4 to generate I(1,2,3,4,6)P5 35. Identified Ipmks include those from human 36 and rat 37.

Human Ipmk is very similar to rat Ipmk with 84% amino acid sequence identity 31. Arabidopsis IP3K and yeast IP3K are less conserved in the Ipmk superfamily with 25% and 16% amino acid sequence identity to human Ipmk respectively 31. Within the catalytic domain of Ipmks family, mammalian homologues share 25-33% and 42-54% identity to yeast and Arabidopsis IP3Ks respectively. Ipmks have conserved IP-binding consensus sequence and ATP-binding site in their catalytic domain 31. Fig. 2 depicts a schematic alignment of IP3Ks from rat, human, yeast and Arabidopsis. The expression patterns of Ipmks are different: rat Ipmk is highly expressed in kidney and brain 37, whereas human Ipmk is ubiquitously expressed 36. Although Arabidopsis IP3K has similar transcript level in flower, root, stem, and leave 15, its activity is detected only in mature pollens, but not in immature pollen grains 15.

Figure 2
figure 2

The structure of IP3K family. The various conserved domains are marked by colored boxes. The dark green box and the blue box show IP3-binding domain and ATP/Mg2+-binding domain, respectively. The red box represents CaM-binding domain. The light green box shows F-actin-binding domain. The SSLL-like motif is also conserved in IP3K. A nucleus localization signal (NLS) is represented by the purple box.

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IP3K STRUCTURE

There are two major functional domains in mammalian IP3Ks: a highly conserved C-terminal catalytic domain and a divergent N-terminal regulatory domain. The structure of mammalian IP3Ks catalytic core (residues 185-459 of rat RnIP33K-A) consists of two domains: a large α/β-class structure and a small α-helical structure 38, 39, 40. The α/β-class structure has two lobes that are necessary for ATP/Mg2+-binding with critical residues Lys-197, Lys-262, Arg-317 and Asp-414, whereas the small α-helical structure is responsible for IP3-binding in dependence of a 35 amino acid sequence of Arg-276 to Lys-303 39, 40, 41, 42, 43. Many hydrophobic residues of the large domain also participate in ATP binding 39. The IP3-binding core is inserted between two lobes of the large domain acting together during ATP binding and phosphate transfer 43, 44. Sequence alignment of IP3Ks shows that consensus sequence PxxxDxKxG is a highly conserved motif for substrate binding 31. However, the small helix domain is absent in mammalian Ipmks, yeast and Arabidopsis IP3Ks 39. This may explain the substrate specificity of IP3 3-kinase and Ipmk from the structure level. Motif [L/M][I/V]D[F/L][A/G][H/K] is also considered as a putative ATP/Mg2+-binding sequence in Ipmks 37. Furthermore Saiardi et al identified a new domain designated “SSLL” in rat Ipmk 37. The SSLL-like motif is also conserved within other IP3Ks 31, 37. Mutational analysis shows that loss of this motif may impair catalysis activity of IP3Ks 37.

Intracellular localization of IP3Ks is contributed by special domains. A novel N-terminal 66 amino acid sequence in rat RnIP33K-A is involved in F-actin binding 45. A similar actin-binding domain was also identified in rat RnIP33K-B 46. Rat RnIP33K-B can bind to ER membrane with high-affinity, depending upon conformation, and protein-protein interaction 30. Soriano and Banting hypothesized that the N-terminus of RnIP33K-B was only required for the binding of the enzyme to the ER in proximity of the IP3 receptor 30. This N-terminal 320 amino acids are unique for the rat IP3K isoform B, which contributes to its subcellular localization to the ER 30. Rat RnIP33K-C is exclusively cytoplasmic but shuttles between cytoplasm and the nucleus 23. A nuclear export signal (NES) has been identified at its N-terminus 23. A similar nuclear localization signal (NLS) has also been discovered in human Ipmk 47. Both yeast and Arabidopsis IP3Ks are nucleus localized 10, 15. However, no obvious NSL can be found through sequence alignment 15. Different domains are presented in Fig. 2.

IP3K REGULATORS

Ca2+/CaM

Mammalian IP3Ks can be activated by CaM in a Ca2+-dependent manner to different degrees. CaM recognizes sequences which contain amphiphilic α-helices with clusters of positively charged and hydrophobic amino acids 38. Sequence from Ser-156 to Leu-189 together with site Trp-165 in rat IP33K-A is required for CaM binding and the enzyme activation 38, 48, 49. The level of stimulation appears to be cell-, tissue- and isoform-specific 27, 50 (Tab. 1). Up to 20-fold of increase in IP3Ks enzymatic activities by Ca2+/CaM can be observed in a in vitro assay using purified IP3Ks from rat 17, 51, pig and human 29, 52, 53. However, IP3Ks from nematode 25, Arabidopsis 15 and yeast 10 lack the consensus CaM-binding sites and thus are insensitive to Ca2+/CaM.

PKA, PKC and CaMKII

Mammalian IP3Ks are substrates of camp-dependent kinase (PKA), protein kinase C (PKC) and Ca2+/CaM-denpendent kinase II (CaMKII). PKA can stimulate IP3K activity. In contrast, PKC is a negative regulator of IP3K 54. Ser-175 on RnIP33K-A is the phosphorylation site for PKC, and Ser-109 for both PKC and PKA 28. Simultaneous phosphorylation of Ser-109 and Ser-175 leads to inactivation of the enzyme, whereas a single phosphorylation at Ser-109 activates it, suggesting that Ser-175 is probably the inhibitory phosphorylation site 28. CaMKII is also a positive regulator of IP3K 55. Thr-311 of human HsIP33K-A is a CaMKII phosphorylation site. CaMKII can stimulate enzyme activity by 8~10-fold 54, 55. The phosphorylation level of IP3K varies depending upon Ca2+/CaM-sensitivity and different isoforms 55. To date, it is not clear whether Ipmk is sensitive to PKC, PKA, CaMKII. But Arabidopsis IP3K can be phosphorylated by PKC in vitro 15. Further experiments are needed to elucidate how the activity of Arabidopsis IP3K is regulated.

Other regulators

Mammalian IP3Ks activity can be stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of cAMP 56, 57, 58. Protein stability is also involved such as mammalian IP3Ks are very sensitive to calpains 59. Pp60v-src kinase can also increase IP3K activity, although the src-phosphorylation site in IP3K has not been identified yet 60.

IP3K FUNCTIONS

IP3Ks are involved in inositol signaling pathway, calcium signal transduction, brain development, stress responses and gene transcription (Fig. 3).

Figure 3
figure 3

The network of IP3K functions.

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Inositol signaling pathway

Mammalian IP3Ks mainly phosphorylate IP3 to IP4 to provide precursors for synthesis of higher IPs 5, 31. Yeast and Arabidopsis IP3Ks participate additional pathway in IP metabolism 10, 15. In yeast, there is a subdivision of lipid-dependent pathway for IP6 synthesis 10. IP3K phosphorylates IP3 stepwise at the D-6 and D-3 positions to generate IP5 or as a minor pathway to phosphorylate IP3 to bring about IP4 10, 14. There is evidence showing that expansion of an IP3 pool could lead to increases of IP4, IP5 and IP6 levels via Ipmk 61. Thus, in higher eukaryotes Ipmk, but not IP3 3-kinase, may be the main contributor for IP5 and IP6 syntheses 61. Plant react in a similar way. Maize IP3K (ZmIpk) is responsible for IP6 biosynthesis in developing maize seed 62. Arabidopsis IP3K has 6-/3-kinase activity and can phosphorylate IP3 to give rise to IP5 15, 35. Besides, Arabidopsis IP3K exhibits a novel 5-kinase activity to produce IP5 from I(1,3,4,6)P4 35. The 5-kinase activity has also been detected in human and Drosophila Ipmks 36, 61, which is especially important for fruit flies since no IP3 5-/6-kinase can be found in this animal. Human Ipmk can also phosphorylate inositol 4,5-biphosphate (IP2) to generate to IP3 and can make pyrophosphate disphosphoinositol tetrakisphosphate (PP-IP4) from IP5 36.

Calcium signal transduction

IP3 and IP4 regulate Ca2+ mobilization synergistically 2, 4. Increase of IP3K activity may reduce cellular IP3 concentration and correspondingly terminate IP3 action. The function of IP4 is implicated in promoting Ca2+ entry from extracellular space 4. Evidence shows that IP4 can activate a protein with ras- and rap-GAP activity and finally inactivate the G protein 30. This indicates that IP4 regulates Ca2+ influx in a GTP-dependent way, which potentially links the IP3 signaling pathway to GTP-regulated signaling mechanisms 30. IP4 is demonstrated to be a common regulator in Ca2+ homeostasis 63. A complete inhibition of IP3K activity in Hela cells by adriamycin or by IP3K-specific antibody blocked Ca2+ oscillations, whereas a partial inhibition caused a significant reduction in oscillations frequency 63. Taken together, IP3K activity is related to the levels of IP3 and IP4 and subsequently to Ca2+ oscillations (Fig. 3). However, it remains unknown whether yeast and Arabidopsis IP3Ks are involved in regulation of Ca2+ oscillations. Recombinant yeast IP3K mainly phosphorylates IP3 to give rise I(1,4,5,6)P4 10, 35. However, I(1,4,5,6)P4 is not as efficient as IP4 in Ca2+ influx. Yeast IP3K thus may not be relevant to Ca2+ oscillations in vivo.

Brain development, memory and learning

Rat and human IP3Ks may be involved in brain development, memory and learning. Rat IP3K activity is low at birth and reaches approximately 50% of adult levels 64. Rat IP3K activities are the highest in the hippocampal CA1 pyramidal neurons, dentate gyrus granule cells, and cerebellar purkinje cells 64, 65. On the other hand, low activities were found in cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts 64, 65. The expression pattern of human HsIP33K-A is similar to that of rat RnIP33K-A. Human HsIP33K-B is predominantly present in astrocytes 66, 67. The distribution of IP3Ks in rat and human brain suggests that IP3K might be involved in brain development and in memory process 68. Spatial learning training leads to the increase of rat RnIP33K-A level , suggesting a possible role of rat RnIP33K-A in spatial learning 69.

Stress responses

Interestingly, a Drosophila IP3K gene (D-IP3K1) appears to be oxidative damage resistant 26. Ubiquitous over-expression of D-IP3K1 confers resistance of flies to H2O2- but not to paraquat-induced oxidative stress 26. Evidence suggests that the protective effect of D-IP3K1 is mainly due to a reduced IP3 level and thus reduced calcium release from internal stores, rather than an increased IP4 level 26. IP3K activity is the key player in this process 26. In yeast, the IP3K activity has also been demonstrated to be required for resistance to salt stress, cell wall integrity and vacuolar morphogenesis 70.

Gene transcription

Yeast IP3K (Ipk2/Arg82) was identified as a regulator of arginine metabonism 10. The complex ArgR-Mcm1 is required to ensure the coordination of gene expression in response to arginine 10, 71. Arg80 and Mcm1 are members of the MADS-box transcription factor family, whereas Arg81, a zinc cluster protein, is the sensor of arginine 72. Three components (Arg80, Arg81 and Mcm1) are sufficient to form a complex with DNA (arginine boxes) in the presence of arginine 72. Yeast IP3K stabilizes Mcm1 and Arg80, and facilitates their assembly into a multimeric complex 72. A poly-Asp domain within amino acid residues 282-303 of yeast IP3K is essential for stability of Arg80 and Mcm1 73. It was argued that the absence of this domain leads to the failure of forming ArgR-Mcm1 transcriptional complex 73. Arabidopsis IP3K has a similar function, which complements yeast Arg82/Ipk2 mutant lacking a functional ArgR-Mcm1 transcriptional complex 15. However, no significant poly-Asp domain is found in Arabidopsis IP3K 15. This evidence is somewhat contradicted to previous hypothesis about the role of yeast IP3K in forming transcriptional complex.

Others

IP4 can bind with high affinity to several intracellular proteins—synaptotagmin (I and II), Gap1, Btk, and centaurin-α—and may interact with synaptotagmin to inhibit synaptic transmission 74. IP4 also acts as a mediator in neuronal death in the ischemic hippocampus 75. The changes in IP3 metabolism may be correlated to critical stages of muscle development and differentiation, which suggests a possible role for IP3K in these processes 76. Moreover, yeast IP3K is involved in cellular mRNA export from the nucleus with Ipk1 and plays a role in determining messenger RNA export from yeast nucleus 10, 77. Recent analysis shows that Arabidopsis IP3K (AtIpk2α) is also associated with pollen germination and root growth 78.

PERSPECTIVE

Signaling crosstalk

IP3K may be a key player in integrating Ca2+ signaling, IP metabolism and other signaling pathways. In plant, Ca2+ levels are modulated by IP3 in response to various signals including hormones, light and abiotic stresses. For example, the addition of abscisic acid (ABA) leads to increase in endogenous IP3 levels 79; red light elicits a rapid Ca2+ intracellular release which can be mimicked by microinjection of IP3 80; gravity stimulates a rapid increase of IP3 in maize 81. However, this may not be the only way for IP3K function in many biological processes. IP4, IP5, and IP6 have been demonstrated functionally important 9, 10, 11, 12, 13, 82. They have recently been implicated as messengers regulating cellular processes including transcription, DNA repair and channel activity 6, 7. IP6 serves as a storage poll of IPs and mineral nutrients in seeds 12, 13. Thus, IP3K may also participate in controlling plant development by regulating subsequent IP signaling pathways. A new exciting function for yeast and Arabidopsis IP3Ks was found in regulation of gene expression 10, 15. A fully understand of the physiological function of IP3K needs a comprehensive consideration of IP3K network. IP3K may simultaneously regulate Ca2+ homeostasis, IP metabolism and gene transcription in response to external stimulus.

Inositol phosphate metabolism

Signals induced by IP3 can be terminated by two ways; either through dephosphorylation by a 5- phosphatase to give inositol 1,4-biphosphate (IP2) or through phosphorylation by IP3K to produce IP4 83. Ipmk may replace IP33-kinase due to their similar enzymatic activities 10, 35. Cellular IP3 serves as a substrate for both IP3 3-kinase and Ipmk to form IP6 61. Ipmk, but not IP33-kinase, is the major enzyme in IP6 synthesis, whereas IP33-kinase mainly function in IP4 synthesis from IP3 61. Different from animal homologues, only two IP3K isoforms (AtIpk2α and AtIpk2β) were isolated from Arabidopsis 15, 35. The general pathway for IP6 synthesis in plant as well as in yeast has been identified as follow: IP3→IP4/I(1,4,5,6)P4→IP5→IP6 35. The first two steps can be phosphorylated by yeast and plant IP3Ks 10, 15, 35. But 3-kinase activity of yeast and Arabidopsis IP3Ks seems less active than their in vivo 6-kinase activity 10, 35. Thus, of particular interest is the mechanism of regulating Ca2+ release and influx in plant cells. However, Arabidopsis IP3K regulating pollen tube growth under different environmental conditions is Ca2+-independent 78.

Gene transcriptional control

The crystal structure of Ipmk is not yet known. Information from mammalian IP3K catalytic domain suggests that yeast and Arabidopsis IP3Ks may interact with other molecules 39, 40. Sun et al copurified COP9 signalsome/CSN from calf brain with inositol 1,3,4-trisphosphate 5/6-kinase 84. This kinase can phosphorylate several transcription factors (NF-κB, c-Jun, p53 etc,) to avoid of degradation by the ubiquitin system 84. Both NF-κB and c-Jun play important roles in brain development and anti-oxidative stress 85, 86. Yeast IP3K activates ArgR-Mcm1 complex and then drives transcription 31. We have also demonstrated that Arabidopsis IP3K complemented ipk2 mutant yeast 15, indicating a potential function of Arabidopsis IP3K in transcription regulation.

IP3K regulation

Mammalian IP3Ks can be modulated by Ca2+/CaM, PKA, PKC, CaMKII and other regulators. However, there is little information about the mechanism. Both yeast and Arabidopsis IP3Ks lack CaM-binding sites and are insensitive to Ca2+/CaM 10, 15. Therefore, compared to mammalian IP3Ks, they are most regulated by different mechanisms. Our preliminary experiments suggest that Arabidopsis IP3K can be phosphorylated by PKC in vitro. Whether such phosphorylation is physiologically relevant to the regulation of Arabidopsis IP3K activity in vivo is not clear. It is important to understand the function regulation of yeast and plant IP3Ks in the near future.