Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

Abstract

Shb is a recently described Src homology 2 (SH2) domain-containing adaptor 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 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated z chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/ 38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation 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 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein–Barr virus LMP2A membrane protein

“…The arrangement of Shb PTB and SH2-binding motifs in LMP2A is strongly reminiscent of a similar motif in CD79a but putative Shb PTB-binding motifs precede the SH2-binding motifs in several ITAM-containing adaptors (Table 1). Previous work implicated PTB-as well as SH2-domain interactions in the binding of Shb to CD3z (Welsh et al, 1998). The finding that LMP2A coopts both Shb and Syk in the regulation of ITAM-mediated activation of the PI3K-Akt pathway may thus exemplify a more general mechanism for regulation of ITAM-mediated signalling in the cell, as the recruitment of Shb to the CD3z during TCR activation suggests (Karlsson et al, 1995;Welsh et al, 1998).…”

“…The fact that Shb is recruited to the CD3z upon TCR stimulation (Welsh et al, 1998) and associates with both SLP76 and Zap70 in T cells (Lindholm et al, 2002) is suggestive of an analogous function in LMP2A-expressing cells.…”

“…Shb associates with tyrosine phosphorylated LMP2A but not with the unphosphorylated N-terminal tail of LMP2A We wished to determine whether the association of Shb with LMP2A might resemble the previously established, phosphorylation-dependent binding of Shb to the CD3z ITAM following TCR stimulation (Welsh et al, 1998). In this experiment we made use of the 3Tm chimeric transmembrane protein where the LMP2A N-terminal signalling domain was fused to the CD38 exodomain (Winberg et al, 2000).…”

“…Previous work implicated PTB-as well as SH2-domain interactions in the binding of Shb to CD3z (Welsh et al, 1998). The finding that LMP2A coopts both Shb and Syk in the regulation of ITAM-mediated activation of the PI3K-Akt pathway may thus exemplify a more general mechanism for regulation of ITAM-mediated signalling in the cell, as the recruitment of Shb to the CD3z during TCR activation suggests (Karlsson et al, 1995;Welsh et al, 1998). In contrast, the presence of proline-rich motifs that bind the WW-domains of Nedd4/AIP4 HECT-domain E3 ubiquitin ligases in close proximity to an ITAM motif appears to be a unique feature of the gamma-herpesviral homologs of LMP2A.…”

“…If so, then such a protein must utilize the LMP2A ITAM motif, which was shown to be required for LMP2A-mediated Akt-activation (Scholle et al, 2000;Swart et al, 2000;Morrison and Raab-Traub, 2005). One such regulatory protein is the signalling scaffold Shb, which is recruited to the ITAM SH2-binding motifs of the CD3z upon T-cell receptor (TCR)-stimulation (Welsh et al, 1998). The proline-rich N-terminal domain of Shb interacts with the Srchomology 3 (SH3) domains of PI3K (Karlsson et al, 1995).…”