| Ganglioside GM3 has been proved to be a physiological cell differentiation inducer in several human leukemia cell lines. However, the inducing differentiation mechanism of GM3 remains unclear. Our previous work has shown that GM3 can induce human monocytoid leukemia J6-2 cell line to differentiate along the monocyte/macrophage route and simultaneously inhibit J6-2 cell phospholipase C ( PLC ) of phosphatidyl inositol ( PI ) specificity. The less activity of PI-PLC can result in the decrease of DAG, which inhibits the activity of PKCα by inhibiting it from the cytosol to the plasma membrane, but the mechanism is still unknown.PI-PLC can hydrolyze phosphatidylinositol 4, 5-bisphosphate ( PIP2 ) into two second messengers, DAG and IP3. About ten isoenzymes of PLC have been found. According to their different structures, PLC can be divided into three subclasses: PLCβ1-4, PLCγ1-2 and PLCδ1-4 . Different PLC subclass has different regulatory mechanism. It is known that family members of PLCβ are activated by G protein coupled receptor ( GPCR ), however, PLC(1 and PLC(2 can be activated by Growth Factor Receptor ( GFR ). In order to study the mechanism of GM3 effect on PLC, the first thing to be settled is to make sure which PLC isoform can be inhibited by GM3. There is no specific method to measure PLC isoform activity in cells. It has been known that PLC( must translocate from the cytosol to the plasma membrane before they were activated, so we examined the translocation of PLC in J6-2 cells affected by GM3, which is helpful for us to study the inhibitory mechanism of GM3 effect on PLC.By releasing the cytosol proteins with Digitonin and centrifugation technology, we divided the J6-2 cell proteins into the cytosol proteins and the particulate-conjugated proteins, and then using SDS-PAGE, Western blotting and Enzyme-linked immunosorbent stain combined with computer quantiscan technology, we investigated the effect of GM3 on PLC(1 translocation. The results showed that the relative content of PLC(1 in the cytosol of J6-2 cells treated with GM3 was higher than that of control cells, whereas the relative content of PLC(1 conjugated with plasma membrane was lower than that of control cells. The results suggested that GM3 inhibited the PLC(1 translocation from the cytosol to the plasma membrane, which signified that the kind of PLC isoform inhibited by GM3 was PLC(1.To locate on the plasma membrane is necessary for PLC to catalyze the hydrolysis of PIP2. PLC(1 can bind to PIP3 by its PH structure and then locate on the membrane. So the content of PIP3 on the membrane is an important molecular for the PLC( location. We use 32P to mark phospholipid on the cell membrane, then use organic solvent abstraction, HPTLC, Autoradiography combined with computer quantiscan technology to detect the effect of GM3 on PIP3 content. The result showed that the content of PIP3 clearly decrease in cells treated with GM3, it is only about 10% of that of blank control. It signified that GM3 inhibit the activity of PI3K obviously and decrease the production of PIP3. This may be the main cause of PLC(1 translocation from cytosol to the plasma membrane inhibited by GM3.PLC( is activated by GFR. Growth Factor binding to receptor ( RPTK) make receptor dimerize and phosphorylate itself, the intracellular domain of receptor produce tyrosine phosphorylation site. PLC( can bind to the tyrosine phosphorylation site of receptors by its SH2 structure and phosphorylated by receptor kinase. We investigated the effect of GM3 on the phosphorylation of GFR ( EGFR, INR, PDGFR ) in J6-2 cells. The results showed the phosphorylation of EGFR treated with GM3 was observably inhibited, GM3 had no obvious effect on other GFR phosphorylation.From the findings above, it was postulated that GM3 could inhibit the activity of PLC(1 possibly via two ways. One is by inhibiting the phosphorylation of EGFR, the other is by decreasing the production of PIP3.
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