Tyrosine residues in phospholipase Cgamma 2 essential for the enzyme function in B-cell signaling.
Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).
Rodriguez
R
R
Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, the Institute of Cancer Research, Fulham Rd., London SW3 6JB, United Kingdom.
Matsuda
M
M
Perisic
O
O
Bravo
J
J
Paul
A
A
Jones
N P
NP
Light
Y
Y
Swann
K
K
Williams
R L
RL
Katan
M
M
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium