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Vol. 62, Issue 3, 672-679, September 2002
2 by Tyrosine Phosphorylation
Departments of Pharmacology (F.Ö., C.D., B.A., S.K., J.L.D.)
and Physiology (S.P.K.) and Sol Sherry Thrombosis Research Center
(C.D., B.A., S.P.K., J.L.D.), Temple University Medical School,
Philadelphia, Pennsylvania
Phospholipase C
2 (PLC2) has been implicated in collagen-induced
signal transduction in platelets and antigen-dependent signaling in
B-lymphocytes. It has been suggested that tyrosine kinases activate PLC2. We expressed the full-length cDNA for human PLC2 in bacteria and purified the recombinant enzyme. The recombinant enzyme
was Ca2+-dependent with optimal activity in the range of 1 to 10 µM Ca2+. In vitro phosphorylation experiments with
recombinant PLC2 and recombinant Lck, Fyn, and Lyn tyrosine kinases
showed that phosphorylation of PLC2 led to activation of the
recombinant enzyme. Using site-directed mutagenesis, we investigated
the role of specific tyrosine residues in activation of PLC2. A
mutant form of PLC2, in which all three tyrosines at positions 743, 753, and 759 in the SH2-SH3 linker region were replaced by
phenylalanines, exhibited decreased Lck-induced phosphorylation
and completely abolished the Lck-dependent activation of PLC2.
Individual mutations of these tyrosine residues demonstrated that
tyrosines 753 and 759, but not 743, were responsible for Lck-induced
activation of PLC2. To confirm these results, we procured a
phosphospecific antibody to a peptide containing phosphorylated
tyrosines corresponding to residues 753 and 759. This antibody
recognized phosphorylated wild-type PLC2 on Western blots but did
not interact with unphosphorylated PLC2 or with PLC2
containing mutated tyrosine residues at 753 and 759. Using this
antibody, we showed in intact platelets that collagen, a
PLC2-dependent agonist, induces phosphorylation of PLC2 at Y753
and Y759. These studies demonstrate the importance of these two
tyrosine residues in regulating the activity of PLC2.
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