Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands.

SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.

The adaptor protein LAT serves as an integration node for signaling pathways that drive T cell activation.

T cells are essential for the adaptive immune response to pathogens. However, dysfunctional T cell activity has been implicated in numerous diseases, including the failure of organ transplants, allergic reactions, asthma, autoimmune disorders, and coronary artery disease. T cell responses to pathogens require the induction of the primary activating receptor, the T cell receptor (TCR), along with other costimulatory and adhesion receptors. Signal transduction pathways activated downstream of these receptors drive T cell responses required for the immune response and disease progression. A key question in our understanding of the mechanism of T cell activation is how signaling pathways emanating from multiple receptors integrate together to alter T cell effector functions. One integration node for intracellular signaling is the membrane-associated adaptor protein linker for the activation of T cells or LAT. Upon stimulation of the TCR and other receptors, LAT is phosphorylated at several tyrosines residues on its cytoplasmic tail. This leads to the binding of SH2 domain-containing proteins and their associated molecules and the formation of large multiprotein complexes. These dynamic and highly regulated signaling complexes facilitate the production of second messengers, activate downstream pathways, induce actin cytoskeleton polymerization, and stimulate the activity of multiple transcription factors. Thus, signaling pathways from several receptors feed into LAT, which then integrates this information and selectively induces pathways critical for T cell activation and the adaptive immune response.

T cell receptor activation leads to two distinct phases of Pyk2 activation and actin cytoskeletal rearrangement in human T cells.

The tyrosine kinase Pyk2 integrates receptor-mediated signals controlling actin cytoskeletal rearrangement, events needed for the activation and function of T cells. Induction of the T cell receptor (TCR) leads to the phosphorylation of Pyk2, but the timing of these events is controversial and not fully understood. In this study, the TCR-induced phosphorylation kinetics of Pyk2 tyrosines 402 and 580 were characterized in human T cells. Interestingly, the early TCR-mediated phosphorylation of Pyk2 was more rapid and transient than ZAP-70, whose phosphorylation kinetics were similar to other T cell signaling proteins. Unexpectedly, Pyk2 had a second burst of phosphorylation 30-60 min after TCR stimulation. Pyk2 was enzymatically active during the two separate bursts of phosphorylation, since both paxillin phosphorylation, a known substrate of Pyk2, and TCR-induced actin polymerization showed similar kinetics. The second burst of Pyk2 phosphorylation did not require actin cytoskeleton rearrangement or PI3K function, but was dependent on the enzymatic activity of Fyn and/or Lck. Collectively, these observations suggest that signaling pathways downstream of TCR activation are hard-wired to induce two separate periods of Pyk2 activation and actin cytoskeletal rearrangement.

Comparison of T cell receptor-induced proximal signaling and downstream functions in immortalized and primary T cells.

BACKGROUND: Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCgamma1, Vav1, and Erk1/Erk2 and substantially more Ca2+ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation. CONCLUSIONS/SIGNIFICANCE: Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line.