STIMULATION THROUGH THE T-CELL RECEPTOR LEADS TO INTERACTIONS BETWEENSHB AND SEVERAL SIGNALING PROTEINS

Shb is a recently described Src homology 2 (SH2) domain-containing ada ptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains, As a result, a number of proteins detected in anti-Shb and anti-Grb2 immun oprecipitates are shared, including phosphoproteins of 22, 36/38, 55/5 7 and 70 kDa. Shb-association with p22, which represents the T cell re ceptor associated zeta chain, occurs through the Shb SH2 domain. The c entral region of Shb binds p36/38. Since this interaction was inhibite d by phosphotyrosine, this region of Shb is likely to contain a non-SH 2 PTB (phosphotyrosine binding) domain. The Shb PTB domain tvas found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library, A peptide corresponding to a phosphorylatio n site in 34 kDa Lnk inhibited association between Shb and p36/38. Ove rexpression of Shb in Jurkat cells led to increased basal phosphorylat ion of Shb-associated p36/38 and p70 proteins, Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crossl inking when expressed in Jurkat cells. Together, our results show thre e distinct domains of Shb all participate in the formulation of multim eric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.