Limited proteolysis of phospholipase C-gamma 1 indicates stable association of X and Y domains with enhanced catalytic activity.
Phospholipase C-gamma 1 (PLC-gamma 1) was treated with Staphylococcus aureus V8 protease (V8) and the digestion products were analysed with site-specific antibodies. V8 treatment generated three immunodetectable PLC-gamma 1 fragments of 120, 97, and 39 kDa. The 39 kDa fragment is derived from the C-terminus of PLC-gamma 1 and includes the conserved Y domain present in all PLC isoenzymes. The 120 and 97 kDa fragments are derived from the N-terminus of PLC-gamma 1, possess the conserved X domain common to all PLC isoenzymes, and the src-homology domains unique to PLC-gamma 1 and -gamma 2. It is likely that the 97 kDa fragment is a V8 product of the 120 kDa fragment. As the C-terminal 39 kDa fragment, and either of the N-terminal 120 or 97 kDa fragments, were precipitable with antibody specific to a sequence present in only the 39 kDa fragment, the data indicate co-precipitation of separate polypeptide chains that remain associated after V8 proteolysis. Importantly, V8 treatment increased the activity of PLC-gamma 1 and did not alter the calcium requirement. The influence of other modulators of PLC-gamma 1 activity, however, was lost following V8 treatment. These results suggest the stable association of the X and Y domains within PLC-gamma 1, and demonstrate that proteolysis in the region of PLC-gamma 1 that is subject to tyrosine phosphorylation can enhance catalytic activity.
Fernald
A W
AW
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146.
Jones
G A
GA
Carpenter
G
G
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments; D051966:Phospholipase C gamma; D011233:Precipitin Tests; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D010446:Peptide Fragments; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase