User:Clairecato/sandbox

Function
Inositol 1,4,5-trisphosphate 3-kinase (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. 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 ITP3K 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 ITP3K important for stabilizing the substrates and products in the active site.

Calcium Signaling Pathway
ITP3K is plays a role in the calcium signaling pathway. In this pathway, either a G-protein coupled receptor (GPCR) or receptor tyrosine kinase (RTK) is activated by an extracellular ligand-binding event. Initiation of the pathway leads to an activated G-alpha subunit of a heterotrimeric G protein (in the case of GPCR-mediated signal transduction) or autophoshorylation of RTK cytoplasmic domains (in the case of RTK-mediated signal transduction). These intracellular events eventually lead to activation of phospholipase C (PLC), which cleaves the phospholipid PIP2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains associated with the plasma membrane, while IP3 is released into the cytoplasm. IP3 then goes on to bind to IP3 receptors on the endoplasmic reticulum or sarcoplasmic reticulum, resulting in an influx of calcium ions into the cytoplasm.

Calcium serves as a second messenger for various downstream cellular events including glycogen metabolism, muscle contraction, neurotransmitter release, and transcriptional regulation. Therefore, calcium homeostasis is essential for proper cell function and response to extracellular signals.

In order to prepare the cell for a future signaling event, the calcium pathway must be tightly regulated. ITP3K seems to play an important role in termination of the signal. As mentioned, ITP3K catalyzes the phosphorylation of IP3 to make IP4. Unlike IP3, IP4 does not cause opening of calcium channels on the endoplasmic reticulum or sarcoplasmic reticulum. By decreasing the concentration of IP3 in the cytoplasm, ITP3K terminates propagation of the calcium signaling pathway.

Additional Roles for ITP3K
ITP3K is not the only enzyme responsible for clearing IP3 from the cytoplasm. A second enzyme called inositol 5-phosphatase catalyzes the dephosphorylation of IP3 to create IP2. Typically, nature does not favor the evolution of a second enzyme to perform an already-existing, identical function. A closer inspection of the evolutionary history of inositol 5-phosphatase and ITP3K gives rise to several interesting hypotheses about the roles of these enzymes in the cell.

Inositol 5-phosphatase existed before ITP3K evolved in the mammalian cell. Like other phosphatases, inositol 5-phosphatase is an energy-independent enzyme that cleaves a phosphate group off of a substrate. In contrast, ITP3K (like all kinases) is energy-dependent, meaning that it requires an ATP molecule to perform the phosphoryl transfer chemistry. If nature already had an energy-independent mechanism for termination of the calcium signaling pathway, why was the evolution of ITP3K advantageous? This apparent redundancy of function, or "waste" of energy by the cell, suggests that ITP3K may have a more important function in the cell than simply clearing the IP3 second messenger from the cytoplasm. Current hypotheses about additional roles for ITP3K are explained in the following two subsections.

The Product of ITP3K may be a Second Messenger
As mentioned previously, ITP3K catalyzes a phosphoryl transfer reaction that converts IP3 to IP4. IP4 does not stimulate calcium influx through IP3 receptor channels on the endoplasmic or sarcoplasmic reticulum. However, it has been shown that IP4 stimulates calcium channel opening on the plasma membrane. In this way, IP4 may actually serve to prolong the calcium signal by activating the influx of calcium stores from the extracellular space.

In addition, there is evidence that IP4 binds two GTPase-activating proteins, GAP1IP4BP and GAP1m. GAPs are often used in signal transduction as on/off switches. IP4 binding to GAPs suggests that ITP3K may be involved in a parallel signal transduction pathway. The exact role of IP4 binding to these GAPs has not been determined, though, so additional research in this area will be needed to gain a more complete understanding.

The Role of ITP3K in Inositol Phosphate Metabolism
In addition to its potential roles as a second messenger, IP4 may also function as an essential precursor for other more highly phosphorylated inositol phosphates such as IP5, IP6, IP7, and IP8. These higher order inositol phosphates are thought to be important for phosphate storage, may serve as additional second messengers, and may be involved in the post-signal recovery phase whereby calcium stores are refilled and the phosphatidylinositol supply is replenished. Such maintenance is necessary to prepare the cell for a future incoming signal.

The generation of the higher order inositol phosphates discussed above relies on the presence of their precursor, IP4. ITP3K is responsible for the formation of IP4. Therefore, it is possible that one of ITP3K’s most important functions is the regulation of IP4 levels, and therefore the regulation of downstream cellular events involving higher order inositol phosphates.

Regulation of ITP3K
ITP3K is regulated by various post-translational mechanisms. The major post-translational modification that is important for ITP3K regulation is phosphorylation. Phosphorylation is one of the most wide-spread protein modifications, and it is especially common in the modulation of signal transduction pathways. ITP3Ks are stimulated directly by calcium/calmodulin (Ca2+/CaM) binding. In addition, 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), but the degrees of these interactions remain elusive.