Inositol (1,4,5) trisphosphate 3-kinase (EC 2.7.1.127), abbreviated here as ITP3K, is an enzyme that facilitates a phospho-group transfer from adenosine triphosphate to 1D-myo-inositol 1,4,5-trisphosphate. This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. The systematic name of this enzyme class is ATP:1D-myo-inositol-1,4,5-trisphosphate 3-phosphotransferase. ITP3K catalyzes the transfer of the gamma-phosphate from ATP to the 3-position of inositol 1,4,5-trisphosphate to form inositol 1,3,4,5-tetrakisphosphate. ITP3K is highly specific for the 1,4,5-isomer of IP3, and it exclusively phosphorylates the 3-OH position, producing Ins(1,3,4,5)P4, also known as inositol tetrakisphosphate or IP4. In biology, the enzyme ITP3K is abbreviated a number of different ways, including 1D-myo-inositol-trisphosphate 3-kinase, ITP3K, ITPK, IP3-kinase, IP3-3-kinase, Ins(1,4,5)P3 3-kinase. In addition the enzyme may be named as the product of one of 3 genes in humans ITPKA, ITPKB, and ITPKC, or one of two in fruit flies, IP3K1 and IP3K2—a mutant known to geneticists as wavy. The nematode genome has one form of the enzyme, coded by the LFE-2 gene. ITP3K enzymes are expressed only in metazoans; they are not expressed in yeast or plants. All ITP3Ks belong to a larger structural family, the inositol polyphosphate kinases, or IPKs. Note however, that the human genome also contains a gene for a different kinase known as ITPK1, which is an inositol 1, 3, 4-trisphosphate 5/6-kinase and is not a member of the IPK family. The ITP3K enzyme family is sometimes confused with a different enzyme family that has a similar name, that is, the phosphatidyl inositol 3-kinases or phosphoinositide 3-kinase (PI3-K), whose substrates are inositol lipids, not the soluble second messenger inositol trisphosphate.
Discovery and characterization Scientific interest in the inositol phosphates intensified in the years following the 1983 discovery that inositol trisphosphate was an intracellular messenger that releases calcium from intracellular stores in the endoplasmic reticulum. By the end of the decade, a large number of inositol phosphate kinases and phosphatases had been discovered, including ITP3K in 1986. Biochemical and molecular studies in the 1990s led to the purification of the enzyme from rat brain and it molecular cloning, and these studies revealed various feedback mechanisms by which the enzyme is regulated by calcium and protein kinases. In 1999, ITP3K was identified as being a member of a larger family of Inositol polyphosphate kinases, which share a similar structure and catalytic mechanism. ITP3K enzymes share common structural features including a conserved catalytic core which binds ATP located near the C-terminus, and various regulatory domains nearer to the N-terminus.
Catalytic domain Evidence for this exquisite specificity and for the catalytic mechanism was found when the apo-enzyme, substrate-bound complex, and product-bound complex X-ray crystal structures of ITPKA were determined. The figure to the right depicts the catalytic mechanism, whereby the 3'OH of IP3 attacks the gamma-phosphate of ATP, and amino acid residues of ITPK important for stabilizing the substrates and products in the active site. The structure of the catalytic domain of the human ITP3KA has been shown to be divided into three subdomains. These subdomains are displayed as the N lobe, which is a N-terminal domain, the C lobe, which is a C-terminal subdomain and a third alpha-only subdomain. The ITP3K catalytic domain varies somewhat from the protein kinase superfamily, and it has a novel four-helix substrate binding domain. In this kinase, the two domains are in an open conformation, which indicates that the two domains are both accessible at the same time. This suggests that substrate recognition and catalysis by ITP3K involves a dynamic conformational cycle. Additionally, this unique helical domain of ITPK blocks access to the active site by membrane-bound phosphoinositides, explaining the structural basis for soluble inositol polyphosphate specificity. Another feature of the catalytic core is the ATP binding site. Here, one molecule of ADP is bound in the cleft of the major domain, which indicates the active site of the kinase. In further detail, the larger domain of the protein structure has an α/β-class structure. The domain has an N-terminal and a C-terminal lobe with a cleft in between and each of these lobes is built around an antiparallel β-sheet. In the N-terminal, the sheet has three strands, whereas in the C-terminal there is a five-stranded sheet. The second domain, is α-helical and consists of four α helices linked by long loops. The helices are loosely packed against each other and the entire domain is highly mobile as compared to the large α/β domain. The helical domain is juxtaposed against one end of the cleft in the large domain.
Regulation ITP3K is regulated by various post-translational mechanisms. ITP3Ks are stimulated directly by calcium/calmodulin (Ca2+/CaM) binding. Generally, mammalian ITP3Ks are activated by calcium and calmodulin to varying degrees. The method in which this works is calmodulin recognizes sequences which contain amphiphilic alpha-helices with clusters of positively charged and hydrophobic amino acids. Certain sequences are required for CaM binding and enzyme activation and this level of stimulation appears to be specific to cell, tissue, or isoform. ITP3Ks from nematodes and Arabidopsis thaliana lack the CaM-binding sites and therefore are insensitive to calcium and calmodulin. Another major post-translational modification that is important for ITP3K regulation is phosphorylation. ITP3K activity is indirectly stimulated by phosphorylation by calcium/calmodulin-dependent kinase II (CaMKII). In addition, there is evidence that ITP3Ks may be activated upon phosphorylation by protein kinase C (PKC) and inhibited upon phosphorylation by protein kinase A (PKA).
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