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Research ArticleArticle

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Fatih Özdener, Carol Dangelmaier, Barrie Ashby, Satya P. Kunapuli and James L. Daniel
Molecular Pharmacology September 2002, 62 (3) 672-679; DOI: https://doi.org/10.1124/mol.62.3.672
Fatih Özdener
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Carol Dangelmaier
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Barrie Ashby
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Satya P. Kunapuli
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James L. Daniel
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Abstract

Phospholipase Cγ2 (PLCγ2) 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 PLCγ2. We expressed the full-length cDNA for human PLCγ2 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 PLCγ2 and recombinant Lck, Fyn, and Lyn tyrosine kinases showed that phosphorylation of PLCγ2 led to activation of the recombinant enzyme. Using site-directed mutagenesis, we investigated the role of specific tyrosine residues in activation of PLCγ2. A mutant form of PLCγ2, 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 PLCγ2. Individual mutations of these tyrosine residues demonstrated that tyrosines 753 and 759, but not 743, were responsible for Lck-induced activation of PLCγ2. 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 PLCγ2 on Western blots but did not interact with unphosphorylated PLCγ2 or with PLCγ2 containing mutated tyrosine residues at 753 and 759. Using this antibody, we showed in intact platelets that collagen, a PLCγ2-dependent agonist, induces phosphorylation of PLCγ2 at Y753 and Y759. These studies demonstrate the importance of these two tyrosine residues in regulating the activity of PLCγ2.

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Molecular Pharmacology: 62 (3)
Molecular Pharmacology
Vol. 62, Issue 3
1 Sep 2002
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Research ArticleArticle

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Fatih Özdener, Carol Dangelmaier, Barrie Ashby, Satya P. Kunapuli and James L. Daniel
Molecular Pharmacology September 1, 2002, 62 (3) 672-679; DOI: https://doi.org/10.1124/mol.62.3.672

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Research ArticleArticle

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Fatih Özdener, Carol Dangelmaier, Barrie Ashby, Satya P. Kunapuli and James L. Daniel
Molecular Pharmacology September 1, 2002, 62 (3) 672-679; DOI: https://doi.org/10.1124/mol.62.3.672
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