Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323.
The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867-8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk. Co-expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-zeta. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-zeta phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.
Lupher
M L
ML
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Rao
N
N
Lill
N L
NL
Andoniou
C E
CE
Miyake
S
S
Clark
E A
EA
Druker
B
B
Band
H
H
D001665:Binding Sites; D001667:Binding, Competitive; D004792:Enzyme Precursors / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000072377:Syk Kinase; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; C000715470:MAS1 protein, human; D010748:Phosphopeptides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C067419:CBL protein, human