Shc Contains Two Grb2 Binding Sites Needed for Efficient Formation of Complexes with SOS in B Lymphocytes

Abstract

Cross-linking of the B-cell antigen receptor (BCR) induces tyrosine phosphorylation of Shc, which is believed to lead to the activation of Ras. Previous work has shown that tyrosine-phosphorylated Shc forms complexes with another adapter protein, Grb2, and the Ras guanine nucleotide exchange factor SOS. Here, we demonstrate that phosphorylation of Shc by the hematopoietic cell-specific tyrosine kinase Syk induces binding of Grb2 to Shc, suggesting that Syk phosphorylates Shc in stimulated B cells. Surprisingly, Syk-phosphorylated Shc possesses two Grb2 binding sites rather than the one site that has been previously reported. Both of these sites are required for efficient formation of Shc-Grb2-SOS complexes in vitro and in vivo. We suggest that two Grb2 proteins anchored by a single Shc protein bind simultaneously to one SOS molecule, resulting in a complex that is more stable than a complex containing only a single Grb2 protein bound to one SOS molecule. This model is consistent with our observation that BCR stimulation greatly increases the amount of SOS associated with Grb2.

SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor

“…9,16 Depending on the cell type and stimulus used, phosphorylation at this site has been proposed to initiate a RAS-independent cascade that culminates, at least, in MYC transcription 9,10 or to reinforce GRB2 binding to SHC to efficiently recruit the SOS GDP/GTP exchange factor. 24 We demonstrate in the present work that activation of the FLT3 kinase in Ba/F3-FF3 cells targets phosphorylation of SHC at tyrosines 239/240 and 313 and that both sites are able to bind GRB2 equally well. GRB2 binding indicates that both tyrosines 239 and 313 are indeed phosphorylated, because phosphorylation of Y240 alone is unable to provide an SH2-binding site for GRB2.…”

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

“…For both B-cell and T-cell antigen receptors, Ras activation seems to be at least partially dependent on tyrosine kinases, and the stimulation of these receptors can lead to the formation of complexes of Shc, Grb2, and/or Sos (31,39,58,60), suggesting that Ras activation can occur through a mechanism similar to the relatively well-characterized recruitment of Sos to receptor tyrosine kinases such as epidermal growth factor receptor. However, Sos has not yet been detected in antigen receptor complexes (8,49).…”

“…All lymphocytes probably express Sos, and this GEF has been implicated in Ras activation occurring downstream of T-cell and B-cell receptors (31,39,58,60). However, in at least some circumstances, T-cell receptor signalling that leads to Ras activation does not seem to result in recruitment of Sos to receptor complexes (8,49).…”

Lymphocyte Antigen Receptor Signal Integration and Regulation by the SHC Adaptor

“…Unauthenticated Download Date | 5/13/18 2:05 AM sponse to BCR aggregation, and as such acquire the capacity to efficiently bind Grb2 (Harper and DeFranco, 1997). Phosphorylation of both sites is required for efficient formation of Shc/Grb2/Sos complexes (Harper and DeFranco, 1997), suggesting a cooperativity of the two Grb2 binding sites in promoting translocation of Sos to the membrane.…”

“…Phosphorylation of both sites is required for efficient formation of Shc/Grb2/Sos complexes (Harper and DeFranco, 1997), suggesting a cooperativity of the two Grb2 binding sites in promoting translocation of Sos to the membrane. According to the model proposed by the authors, this would be achieved by two Grb2 molecules simultaneously binding to a single Sos molecule.…”