Activation of phospholipase Cgamma2 by tyrosine phosphorylation.
Phospholipase Cgamma2 (PLCgamma2) 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 PLCgamma2. We expressed the full-length cDNA for human PLCgamma2 in bacteria and purified the recombinant enzyme. The recombinant enzyme was Ca(2+)-dependent with optimal activity in the range of 1 to 10 microM Ca(2+). In vitro phosphorylation experiments with recombinant PLCgamma2 and recombinant Lck, Fyn, and Lyn tyrosine kinases showed that phosphorylation of PLCgamma2 led to activation of the recombinant enzyme. Using site-directed mutagenesis, we investigated the role of specific tyrosine residues in activation of PLCgamma2. A mutant form of PLCgamma2, 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 PLCgamma2. Individual mutations of these tyrosine residues demonstrated that tyrosines 753 and 759, but not 743, were responsible for Lck-induced activation of PLCgamma2. 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 PLCgamma2 on Western blots but did not interact with unphosphorylated PLCgamma2 or with PLCgamma2 containing mutated tyrosine residues at 753 and 759. Using this antibody, we showed in intact platelets that collagen, a PLCgamma2-dependent agonist, induces phosphorylation of PLCgamma2 at Y753 and Y759. These studies demonstrate the importance of these two tyrosine residues in regulating the activity of PLCgamma2.
Ozdener
Fatih
F
Department of Pharmacology, Temple University Medical School, Philadelphia, Pennsylvania 19140, USA.
Dangelmaier
Carol
C
Ashby
Barrie
B
Kunapuli
Satya P
SP
Daniel
James L
JL
D001792:Blood Platelets / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011994:Recombinant Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma