Hypophosphorylated TCR/CD3zeta signals through a Grb2-SOS1-Ras pathway in Lck knockdown cells.
Despite the loss of proximal TCR-dependent signaling events, downstream T cell responses are paradoxically augmented in T cells with siRNA-mediated Lck knockdown (Methi et al., J. Immunol. 2005. 175: 7398-7406). This indicates that alternative Lck-independent pathways of T cell activation exist or that low levels of Lck elicit other signals than normal T cell activation. Here we report the recruitment of Grb2-SOS1 to CD3zeta of the TCR complex after prolonged anti-CD3 (OKT3) stimulation in T cells with Lck knockdown. Grb2 bound to incompletely phosphorylated ITAM1 with the pY-Y configuration in a solid-phase assay, but was excluded by ZAP-70 in the doubly phosphorylated pY-pY conformation. Ras and ERK1/2 activation was augmented after prolonged stimulation in T cells with Lck knockdown compared to control, leading to increased activation of the proximal IL-2 promoter (NFAT-AP-1). Finally, the phosphorylation of Ras-GAP was strongly suppressed in Lck knockdown cells, indicating that a Ras negative feedback mechanism is dependent on Lck.
Methi
Trond
T
The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Ngai
Jacob
J
Vang
Torkel
T
Torgersen
Knut M
KM
Taskén
Kjetil
K
D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D020837:SOS1 Protein / Q000378:metabolism*; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D051380:GRB2 Adaptor Protein; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; D020837:SOS1 Protein; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D018631:ras Proteins