Grb2 Forms an Inducible Protein Complex with CD28 through a Src Homology 3 Domain-Proline Interaction

Abstract

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52 shc bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28. Engagement of the T cell receptor (TCR)1 by the major histocompatibility complex-peptide complex in the absence of costimulatory molecules is insufficient to induce production of cytokines and can render the T cells unresponsive to further antigenic challenge (1). CD28 is preeminent among a group of receptors, including 4 -1BB and CD43, that can provide costimulatory signals to T cells (2-4). CD28 is a type 1 transmembrane protein of the Ig superfamily, which is expressed on the cell surface as a glycosylated homodimer (5). CD28 costimulation of TCR-dependent responses increases IL-2 production (6, 7), prevents the induction of anergy (8), and renders T cells resistant to apoptotic cell death (9 -11). These effects are mediated by increased transcription of cytokine genes through the activation of a composite NF-B and AP-1 transcriptional element (12, 13), the stabilization of cytokine mRNAs (14), and the expression of the survival protein, Bclx L (9). TCR-mediated T cell activation is sensitive to the immunosuppressive drug cyclosporin, while those pathways activated by CD28 are not, suggesting that costimulatory pathways are distinct from those activated by the TCR (7). Early biochemical events induced through CD28 include tyrosine phosphorylation (15, 16) and activation of PI3K (17-19) and acidic sphingomyelinase (20). The identity of the signal transduction pathways that are required for CD28-mediated costimulation are presently unknown.The cytoplasmic domain of CD28 contains no recognized intrinsic enzymatic activity; however, CD28 has been reported to associate with signaling proteins following ligation. Phosphorylation of CD28 on tyrosine 173 within the motif YMNM pres...

SH2-B, a Membrane-associated Adapter, Is Phosphorylated on Multiple Serines/Threonines in Response to Nerve Growth Factor by Kinases within the MEK/ERK Cascade

“…In addition to its SH2 domain, it has multiple proline-rich motifs, a PH domain, and multiple potential serine, threonine, and tyrosine phosphorylation sites. PH domains and proline-rich motifs are present in many signaling molecules and are thought to target these proteins to the plasma membrane by binding to phospholipids (50 -52) and constitutively associate with SH3 or WW domain-containing proteins, respectively (53)(54)(55)(56)(57)(58)(59). Therefore, we think it likely that its PH domain targets SH2-B to the plasma membrane, and its proline-rich motifs interact constitutively with signaling molecules containing SH3 domains.…”

CD28 co‐signaling in the adaptive immune response

“…At the same time, a distal proline-rich P 187 YAP region has been implicated. This proline-rich site has been reported to bind to the SH3-domain of src kinase p56 lck , Grb2 and filamin A (FLNa) 61,76,94,95 (Fig. 4).…”

“…In 1995, Schneider et al showed that the CD28 YMNM motif can also bind to the adaptor protein Grb2, by virtue of the YXNX sequence, 49,61,76 where X denotes any amino acid residue (Figs. 4 and 5).…”

“…Grb2 consists of one SH2 domain that is flanked by two SH3 domains, which react with the PYAP motif of CD28 as well. 61,76 Grb2 in turn constitutively binds a guanosine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1), which activates p21 ras and the MAPK/ERK pathway. The loss of Grb2 binding by mutation of the asparagine (N) residue leads to a loss of CD28 mediated phosphorylation of the guanine nucleotide exchange factor Vav1 and consequent activation of the serine/threonine kinase c-Jun kinase (JNK).…”

Induction of autoimmune disease in CTLA-4−/−mice depends on a specific CD28 motif that is required forin vivocostimulation

“…The cytosolic tail of CD28 consists of 41 aa and lacks intrinsic catalytic activity but contains distinct binding motifs that serve as docking sites for different intracellular kinases whose activity may be enhanced by binding to the CD28 cytosolic domain. In vitro molecular studies with recombinant proteins have demonstrated that the ''YMNM motif'' from residues 170-173 binds phosphoinositide 3-kinase (PI3K), Grb2, and Gads; the ''N-terminal proline'' motif from residues 175-178 binds IL2-inducible T cell kinase (Itk); and the ''C-terminal proline motif'' at residues 187-190 binds lymphocyte-specific protein tyrosine kinase (Lck), Fyn, and Grb2 (3)(4)(5)(6)(7)(8)(9)(10)(11). Despite much effort, it has been difficult to clearly identify which CD28 binding motifs are responsible for CD28 costimulatory functions, because most attempts at answering this question have yielded complex and contradictory results.…”