Phospholipases

A) The hypothesis being tested here is the enhancement in the lipase activity of phospholipase C-?1 via phosphorylation of tyrosine 783.B) To perform the experiment equal concentrations of purified phospholipase-C-?1 were set on incubation with the active kinase domain of FGFR2 and ATP in bovine serum albumin containing buffer. The samples of this reactions were tested for two activities: 1) for lipase activity in the phospholipid vehicles indicated in the figure on left y axis. Secondly the phosphate incorporation in phospholipase-C-?1 was studied, illustrated at right y axis of figure.This was performed to check the phosphorylation of tyrosine and auto inhibition of PLC-? isozymes, 775/783 of PLC-?1 were substitutes at the place of phenylalanine, they could be used individually or together, but in the experiment tyr783 is used individually. Phospholipase activity of resulting mutant after purification was quantified with active domain of FGFR2K (helps in phosphorylation and activation of phospholipase). certain known moles of phosphates were added into purified PLC-?1 in wild type under above mentioned conditions and was observed that phospholipase activity was enhanced 10 times. The mutation of tyr783 completely nullified the kinase stimulated acceleration of phospholipase activity along with reduction in FGFR2K-promoted phosphorylation of PLC-?1. Therefore, phosphorylation of Tyr783 is vital forrelief of auto-inhibition. C) Studies reveal that Tyr-783 was essential for auto inhibition. As discussed above, permanent phosphorylation of tyr-783 will completely nullify the kinase stimulated and FGFR2K stimulated phosphorylation of PLC-?1. lipase activity of PLC-?1 will be enhanced across its limits and over-expression of PLC-?1 can induce malignant transformation. The results could be leading to production of carcinoma cells. It has been found in various studies that activity of PLC-?1 is more in cancerous cells as compared to normal cells. So, permanent phosphorylation tyr783 could be a way leading to malignant cancers.