SLP-65 signal transduction requires Src homology 2 domain-mediated membrane anchoring and a kinase-independent adaptor function of Syk.
The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Abulizi
A
Abudula
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Annika
A
Grabbe
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Markus
M
Brechmann
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Christian
C
Polaschegg
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Nadine
N
Herrmann
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Ingo
I
Goldbeck
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.
Kai
K
Dittmann
Georg August University of Göttingen, Institute of Cellular & Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany. Electronic address: jwienan@uni-goettingen.de.
Jürgen
J
Wienands
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D000072377:Syk Kinase; D013601:T-Lymphocytes / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; C113939:B cell linker protein; D047908:Intracellular Signaling Peptides and Proteins; C110810:LAT protein, human; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D010750:Phosphoproteins; C092947:SLP-76 signal Transducing adaptor proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D002118:Calcium