Removal of the carboxyl-terminal region of phospholipase C-beta 1 by calpain abolishes activation by G alpha q.
The 150-kDa phospholipase C (PLC)-beta 1 and three immunologically related proteins with molecular sizes of 140, 100, and 45 kDa were purified from bovine brain extracts. Determination of the amino-terminal amino acid sequence of the 45-kDa protein and immunoblots of the purified proteins with sequence-specific antibodies to peptides corresponding to three different regions of PLC-beta 1 suggest that a single cleavage at the linkage between amino acid residues 880 and 881 of PLC-beta 1 generates the 100- and 45-kDa proteins, which correspond to the amino-terminal and carboxyl-terminal portions, respectively, of PLC-beta 1. The Ca(2+)-dependent protease calpain appears to be responsible for the cleavage of PLC-beta 1; the PLC-beta 1 amino acid sequence contains PEST sequences which are common to proteins susceptible to calpain, and limited proteolysis of purified PLC-beta 1 by calpain generated a 100-kDa protein and a 40-kDa protein that contains the same amino-terminal sequence as the 45-kDa protein. The 140-kDa protein lacks the carboxyl-terminal-most region of PLC-beta 1, but there is no evidence it is derived from PLC-beta 1 by proteolysis. Cleavage of PLC-beta 1 by calpain had no significant effect on catalytic activity measured in the absence of the alpha subunit of the G alpha q but completely abrogated the stimulatory effect of G alpha q. On the other hand, G alpha q activated the 140-kDa enzyme. These results suggest that the region between residue 881 and the most carboxyl-terminal 10 kDa of PLC-beta 1 contains the G alpha q interaction site.
Park
D
D
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Jhon
D Y
DY
Lee
C W
CW
Ryu
S H
SH
Rhee
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology*; D002154:Calpain / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D007527:Isoenzymes; D010455:Peptides; D010738:Type C Phospholipases; D002154:Calpain; D019204:GTP-Binding Proteins