Requirement for a negative charge at threonine 60 of the FcRgamma for complete activation of Syk.
Aggregation of FcepsilonRI on mast cells results in the phosphorylation of the FcepsilonRIgamma chain on tyrosine and threonine residues within the immunoreceptor tyrosine-based activation motif. In the present study we sought to identify the site of threonine phosphorylation in FcepsilonRIgamma and investigate its functional importance. We found that threonine 60 was phosphorylated in vitro and in vivo. Expression of a mutated FcepsilonRIgamma (T60A), in either FcepsilonRIgamma-deficient or gamma-null mast cells, resulted in a delay of FcepsilonRI endocytosis, inhibition of TNF-alpha mRNA production, and inhibition of degranulation but did not affect FcepsilonRI-induced cell adhesion. Tyrosine phosphorylation of the T60A mutant gamma chain was normal, but Syk phosphorylation was dramatically reduced in these transfectants. This correlated with reduced co-immunoprecipitation of FcepsilonRIgamma with Syk. Substitution of an aspartic residue for threonine 60 of the FcepsilonRIgamma reconstituted complete activation of Syk and co-immunoprecipitation of FcepsilonRIgamma with Syk. We conclude that the negative charge provided by phosphorylation of threonine 60 of the FcepsilonRIgamma is required for the appropriate interaction and activation of Syk. This is a likely requirement for immunoreceptor tyrosine-based activation motifs involved in Syk activation.
Swann
P G
PG
Section on Chemical Immunology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Maryland 20892, USA.
Odom
S
S
Zhou
Y J
YJ
Szallasi
Z
Z
Blumberg
P M
PM
Draber
P
P
Rivera
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005786:Gene Expression Regulation; D047908:Intracellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D000072377:Syk Kinase; D013912:Threonine / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D017931:DNA Primers; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D017455:Receptors, IgE; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat