Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.
Holland
S J
SJ
Rigel, Incorporated, South San Francisco, CA 94080, USA. sholland@rigel.com
Liao
X C
XC
Mendenhall
M K
MK
Zhou
X
X
Pardo
J
J
Chu
P
P
Spencer
C
C
Fu
A
A
Sheng
N
N
Yu
P
P
Pali
E
E
Nagin
A
A
Shen
M
M
Yu
S
S
Chan
E
E
Wu
X
X
Li
C
C
Woisetschlager
M
M
Aversa
G
G
Kolbinger
F
F
Bennett
M K
MK
Molineaux
S
S
Luo
Y
Y
Payan
D G
DG
Mancebo
H S
HS
Wu
J
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics* / Q000276:immunology*; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000276:immunology*; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D000099192:CD69 Antigens
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D002118:Calcium; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D037181:Lectins, C-Type; D019814:Myristic Acid; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016392:Proto-Oncogene Proteins pp60(c-src); D020794:Receptor Protein-Tyrosine Kinases; D011947:Receptors, Antigen, B-Cell; D011948:Receptors, Antigen, T-Cell; D013752:Tetracycline; D015534:Trans-Activators; D014157:Transcription Factors; D014443:Tyrosine; D000099192:CD69 Antigens; C490635:SLA protein, human; C441328:SLA2 protein, human; C486695:Sla2 protein, mouse; C084944:TYRO3 protein, human