Abstract
The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.
Highlights
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The expression of mutant Gads with an inactivated Src homology 2 (SH2) domain (SH2*) appreciably reduced tyrosine phosphorylation of hemopoietic progenitor kinase-1 (HPK1), suggesting that Gads may be directly involved in coupling HPK1 to tyrosine kinases that are activated by the TCR
Our studies reveal that a small proportion of HPK1 becomes rapidly tyrosinephosphorylated following TCR activation
Summary
SH, Src homology; PLC, phospholipase C; Gads, Grb2-related adaptor downstream of Shc; Grap, Grb2-related adaptor protein; SAPK, stress-activated protein kinase; GCK, germinal center kinase; GLK, GCK-like kinase; GCKR/KHS, GCK-related kinase/kinase homologous to SPS1/Ste; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; LAT, linker of activated T cells; HPK1, hemopoietic progenitor kinase 1; mHPK1, murine HPK1; pY, phosphotyrosine; HA, hemagglutinin; PVDF, polyvinylidene difluoride; MBP, myelin basic protein. Grb2-Sos complexes to tyrosine-phosphorylated LAT, a membrane-bound linker protein [14, 15, 36]. Grap may function to amplify TCR activation of Ras through recruitment of additional pools of Sos to membrane-associated LAT. We show that HPK1 is activated by the TCR, that Gads and HPK1 interact in vivo, and that Gads can function to link HPK1 to signaling from the activated TCR complex
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