T-cell development in the thymus is a complex and ordered process. Precursor cells that commit to the T-cell lineage fate develop into either the αβ or γδ lineage (12
). Several steps in the maturation of αβ T cells in the thymus have been recognized. Immature thymocytes that do not express either CD4 or CD8 surface molecules (termed double negative [DN]) progress to express both CD4 and CD8 concurrently (double positive [DP]). DP thymocytes then differentiate into cells expressing either CD4 or CD8 (single positive [SP]).
The DN thymocytes can be further subdivided into at least four distinct developmental stages (DN1, DN2, DN3, and DN4) based on the differential surface expression of CD25 and CD44 molecules: CD25−
). Recent studies suggest further subpopulations within the DN1 subset (26
). Recombination at the T-cell receptor β (TCR-β) locus is initiated during the DN2 stage. Successful rearrangement at the TCR-β locus results in expression of TCR-β protein, which forms a heterodimer with the pre-Tα chain and appears on the surface as a pre-TCR. Signaling via the pre-TCR at the DN3 stage (termed “β selection”) provides cues for maturation to the DN4 and DP stages. It has not been established whether DN4 represents a transitional cell type on its way to becoming DP or a cell type that receives/responds to signals via the pre-TCR and additional cues.
Phosphorylation and activation of the serine/threonine kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2 (collectively referred to here as ERK) have been shown to be important for DN thymocyte maturation. Inhibition of ERK phosphorylation via drugs that affect upstream kinases (Mek1) or dominant negative forms of Mek1 blocks thymocyte maturation at the DN3 stage (5
). Moreover, forced expression of activated Ras or activated Raf, which leads to ERK activation, promotes maturation of DN thymocytes to the DP stage in the pre-TCR deficient RAG−/−
). A recent study in which both erk1
genes were deleted within thymic subpopulations revealed a key role for ERK1/2 proteins at the β selection checkpoint (7
). That study and other studies suggest a role for ERK at least through the DN4 stage of development.
Activation of the small GTPase Ras and initiation of a Ras-mediated kinase cascade lead to phosphorylation of ERK on specific threonine and tyrosine residues and its subsequent activation. In mature T cells, several adapter proteins have been implicated upstream of Ras/ERK activation (3
). These include the adapters LAT, SLP-76, GADS, Shc, and Grb2. Both LAT and Shc can recruit Grb2 and in turn Sos, a Ras guanine nucleotide exchange factor, and thereby promote Ras/ERK activation. LAT and SLP-76, through their role in calcium flux via phospholipase C-γ1 (PLC-γ1) can also lead to protein kinase C activation and in turn to downstream activation of a second Ras guanine nucleotide exchange factor, Ras-GRP1 (6
). The importance of the contributions of these different adapters in regulating ERK activation during thymic development has not been defined. While ablation of LAT, ShcA, and SLP-76 expression in mice results in arrested thymocyte development at the DN3 stage, how they link to ERK phosphorylation in DN thymocytes is not known (1
). Ras-GRP1 expression is low in DN cells prior to pre-TCR signaling, and Ras-GRP1 null mice do not have a demonstrable defect in the DN→DP transition (6
). Thus, the relative contributions of the different adapters in regulating ERK phosphorylation in DN thymocytes constitute an important yet unanswered question.
ShcA (encoded by the shc1
gene) is a ubiquitously expressed adapter protein (16
) that contains an N-terminal phosphotyrosine-binding (PTB) domain, a central proline-rich (CH1) domain, and a C-terminal Src homology 2 (SH2) domain (16
). Both the SH2 and PTB domains of ShcA can bind phosphorylated tyrosine motifs. In T-cell lines, ShcA can be recruited to the TCR complex either via the Shc-SH2 domain binding to phosphorylated TCR-ζ chain (28
) or via the Shc-PTB domain binding to tyrosine-phosphorylated ZAP-70 (20
) or Lck (8
). In DN thymocytes (and in mature T cells), ShcA becomes phosphorylated on three tyrosine residues, Y239, Y240, and Y317, within the CH1 domain. This in turn provides a binding motif for SH2-containing proteins, such as Grb2 (25
). The recruitment of Grb2:Sos and the subsequent activation of the Ras/ERK pathway has been implicated as one mechanism of Shc-mediated downstream signaling (32
). A ShcA protein carrying mutations of all three tyrosines (ShcFFF) is nonfunctional and has a potent dominant negative effect in T-cell activation (21
Here we report that ShcA is required for up to 70% of ERK phosphorylation in DN thymocytes. Using mice lacking ShcA expression or mice expressing tyrosine mutants of ShcA, we demonstrated that ERK activation critically requires ShcA protein expression as well as two specific tyrosine residues on ShcA.