Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker.
Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.
Humphries
Lisa A
LA
Molecular Biology Institute and Department of Microbiology and Immunology, UCLA, Los Angeles, California 90095, USA.
Dangelmaier
Carol
C
Sommer
Karen
K
Kipp
Kevin
K
Kato
Roberta M
RM
Griffith
Natasha
N
Bakman
Irene
I
Turk
Christoph W
CW
Daniel
James L
JL
Rawlings
David J
DJ
D000595:Amino Acid Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D014443:Tyrosine; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium