| Phospho‑SLP‑76 (Ser376) represents a negative‑feedback state of the TCR scaffold SLP‑76 in which HPK1‑driven serine phosphorylation converts this otherwise activating adaptor into a platform for 14‑3‑3 binding, ubiquitination, and proteasomal degradation, thereby dismantling LAT/SLP‑76 signalosomes and attenuating TCR signaling intensity and duration. SLP‑76 contains N‑terminal acidic and tyrosine motifs, a central proline‑rich region, and a C‑terminal SH2 domain that together orchestrate assembly of ZAP‑70–phosphorylation–dependent complexes with LAT, Vav, Nck, Itk, and PLCγ1 after TCR engagement; Ser376 lies in the C‑terminal portion of the adaptor and forms part of a canonical 14‑3‑3 recognition motif when phosphorylated. Following TCR ligation, HPK1 (MAP4K1), which is recruited to the LAT/SLP‑76 complex, phosphorylates SLP‑76 on Ser376 in a kinase‑activity–dependent manner, and this modification is required for docking of 14‑3‑3 adaptor proteins such as 14‑3‑3σ and 14‑3‑3ε onto SLP‑76, as shown by structural and biophysical characterization of a Ser376‑phosphopeptide bound within the amphipathic groove of 14‑3‑3. Ser376 phosphorylation not only promotes 14‑3‑3 binding but also triggers Lys30‑linked ubiquitination of SLP‑76 and its proteasomal degradation during ongoing TCR signaling; mutation of Ser376 or Lys30 reduces SLP‑76 ubiquitination, stabilizes the adaptor, and leads to enhanced and prolonged ERK and JNK activation in response to CD3 stimulation, demonstrating that this serine phospho‑switch directly tunes MAPK output downstream of the TCR. Disruption of HPK1 or pharmacologic HPK1 inhibition markedly diminishes SLP‑76 Ser376 phosphorylation in Jurkat and primary human CD8⁺ T cells and is associated with sustained ERK phosphorylation, increased IL‑2 production, and augmented T‑cell activation, and genetic or biochemical evidence indicates that the citron homology domain of HPK1 contributes to SLP‑76 docking and efficient Ser376 phosphorylation. |
