A double point mutation in PCL-γ1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Abstract

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-γ1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-γ1, abolished interactions with translational elongation factor 1-α. Here, we report that the Y509A/F510A mutant PLC-γ1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-γ1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-γ1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-γ1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-γ1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-γ1 activation in vivo.

Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML

“…At first, we found higher peptide phosphorylation of PLC-γ1 at a tyrosine residue Y783 relative to NBM and CN-AML which was opposite to PLC-γ1 serine residue (S1248). PLC-γ1_Y783 is known to be a critical phosphorylation site for PLC-γ1 enzymatic activation whereas PLC-γ1_S1248 negatively regulates PLC-γ1 activity [ 23 , 24 ]. The PLC-γ1 peptide phosphorylation was confirmed at protein levels both phosphorylation and total protein of PLC-γ1 in primary t(8;21) AML samples and kasumi-1 cells compared to other karyotypes and cell lines.…”

“…PLC-γ1 can be phosphorylated at tyrosine residues (Y783, Y771) and also at a serine residue (S1248). Among these residues, PLC-γ1_Y783 is known to be a critical phosphorylation site for PLC-γ1 enzymatic activation whereas PLC-γ1_S1248 negatively regulates PLC-γ1 activity [ 23 , 24 ]. PLC-γ1 is a key signaling molecule that hydrolyzes phosphatidylinositol-4,5-biophosphate to generate inositol-1,4,5-triophosphate (IP3) and 1,2-diacylglycerol (DAG), which in turn, activate intracellular Ca 2+ and protein kinase C (PKC) signaling pathways [ 25 ].…”

Phosphatidylinositol phosphates directly bind to neurofilament light chain (NF-L) for the regulation of NF-L self assembly

“…(Suh et al, 1988;Berridge, 1993;Rhee, 2001;Shin et al, 2002). PLC-γ1 has two pleckstrin homology (PH) domains for protein-protein and proteinlipid interactions (Gibson et al, 1994;Falasca et al, 1998;Chung et al, 2010): PH1 is located in the 150 amino acid residues closest to the N-terminus, while PH2 is located near the center of the molecule and is split by an SH2-SH2-SH3 domain (Gibson et al, 1994;Chang et al, 2002). PH domains bind with high specificity and affinity to phosphatidylinositol phosphates (PtdInsPs) such as PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 (Lemmon and Ferguson, 2000;Cozier et al, 2004;Carlton and Cullen, 2005;Balla, 2005).…”