• Journal of fish pathology
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• v.20 no.2
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• pp.119-127
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• 2007

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to characterize the catalytic and regulatory properties of the PLCδ of Misgurnus mizolepis (ML-PLCδ). The ML-PLCδ gene was cloned and expressed under according to the method of the previous report (Kim et al., 2004), and its recombinant protein was purified by successive chromatography using Ni2+-NTA affinity column. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependence, which was similar to mammalian PLCδ isozymes. The Ca2+ concentration yielding maximal activation of ML-PLCδ was 100 μM. However, the activity was decreased interestingly by a polyamine, such as spermine and spermidine. In vitro assay using cholate-micelle cell, ML-PLCδ activity was inhibited in dose-dependent manner by sphinogosine but increased by phosphocholine . In the lipid-binding assay, ML-PLCδ was strongly bound to LPA, PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA, but it showed the low affinity to S1P, PI(3,4)P2 and PS. Taken together our results, it is suggested that the general catalytic and regulatory properties of ML-PLCδ are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended piscine phospholipase Cδ1 (ML-PLCδ) might reflect some distinctions in regulatory properties and inositol-lipid binding specificity between piscine ML-PLCδ and mammalian PLCδ isozymes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Journal of Life Science
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• v.16 no.4
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• pp.612-617
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• 2006

Phosphoinositide-specific phospholipase $C-{\gamma}\;1\; (PLC-{\gamma}\;1)$ has pivotal roles in cellular signaling by producing second messengers, inositol 1,4,5-trisphosphate $(IP_3)$ and diacylglycerol (DG). Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of ${\alpha}-$ and ${\beta}-tubulin$ heterodimers in all eukaryotic cells. In humans, six ${\beta}-tubulin$ isotypes have been identified which display a distinct pattern of tissue expression. Previously we found that $PLC-{\gamma}\;1$ and one of four ${\beta}-tubulin$ isotypes including ${\beta}1$, ${\beta}2$, ${\beta}3$ and ${\beta}6$, colocalized in COS-7 cells and cotranslocated to the plasma membrane to activate $PLC-{\gamma}\;1$ upon agonist stimulation. In the present study, we demonstrate that the remaining two, tubulin ${\beta}4$ and ${\beta}5$, also showed a potential to activate $PLC-{\gamma}\;1$. The phosphatidylinositol 4,5-bisphosphate $(PIP_2)$ hydrolyzing activity of $PLC-{\gamma}\;1$ was substantially increased in the presence of purified ${\beta}4$ and ${\beta}5$ tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that tubulin ${\beta}4$ and ${\beta}5$ also activate $PLC-{\gamma}\;1$. Taken together, our results suggest that all the ${\beta}-tubulin$ isotype activates $PLC-{\gamma}\;1$ activity to regulate cellular signaling.

• Journal of Life Science
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• v.30 no.9
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• pp.826-833
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• 2020

Phospholipase C gamma (PLCγ) has critical roles in receptor tyrosine kinase- and non-receptor tyrosine kinase-mediated cellular signaling relating to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] to produce inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG), which promote protein kinase C (PKC) and Ca²⁺ signaling to their downstream cellular targets. PLCγ has two isozymes called PLCγ1 and PLCγ2, which control cell growth and differentiation. In addition to catalytically active X- and Y-domains, both isotypes contain two Src homology 2 (SH2) domains and an SH3 domain for protein-protein interaction when the cells are activated by ligand stimulation. PLCγ also contains two pleckstrin homology (PH) domains for membrane-associated phosphoinositide binding and protein-protein interactions. While PLCγ1 is widely expressed and appears to regulate intracellular signaling in many tissues, PLCγ2 expression is restricted to cells of hematopoietic systems and seems to play a role in the regulation of immune response. A distinct mechanism for PLCγ activation is linked to an increase in phosphorylation of specific tyrosine residue, Y783. Recent studies have demonstrated that PLCγ mutations are closely related to cancer, immune disease, and brain disorders. Our review focused on the physiological roles of PLCγ by means of its structure and enzyme activity and the pathological functions of PLCγ via mutational analysis obtained from various human diseases and PLCγ knockout mice.