RhoA activates purified phospholipase C-epsilon by a guanine nucleotide-dependent mechanism.
Phospholipase C-epsilon (PLC-epsilon) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (Galpha(12), Galpha(13), and Gbetagamma) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-epsilon as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-epsilon encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; approximately 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-beta isozymes, the purified fragment of PLC-epsilon maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of approximately 10 mumol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-epsilon fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique approximately 65 amino acid-insert within the catalytic core of PLC-epsilon. Although Rac1 activated purified PLC-beta2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-epsilon. These results indicate that PLC-epsilon is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC-epsilon depends on a unique insert within the catalytic core of the phospholipase.
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Jason P
JP
Seifert
Michele R
MR
Wing
Jason T
JT
Snyder
Svetlana
S
Gershburg
John
J
Sondek
T Kendall
TK
Harden
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D006150:Guanine Nucleotides; D007527:Isoenzymes; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; C037178:phosphatidylinositol 4-phosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D002118:Calcium