Phosphorylation of CARMA1 by HPK1 is critical for NF-κB activation in T cells

Abstract

Activation of the NF-κB pathway in T cells is required for induction of an adaptive immune response. Hematopoietic progenitor kinase (HPK1) is an important proximal mediator of T-cell receptor (TCR)-induced NF-κB activation. Knock-down of HPK1 abrogates TCR-induced IKKβ and NF-κB activation, whereas active HPK1 leads to increased IKKβ activity in T cells. Yet, the precise molecular mechanism of this process remains elusive. Here, we show that HPK1-mediated NF-κB activation is dependent on the adaptor protein CARMA1. HPK1 interacts with CARMA1 in a TCR stimulation-dependent manner and phosphorylates the linker region of CARMA1. Interestingly, the putative HPK1 phosphorylation sites in CARMA1 are different from known PKCθ consensus sites. Mutations of residues S549, S551, and S552 in CARMA1 abrogated phosphorylation of a CARMA1-linker construct by HPK1 in vitro. In addition, CARMA1 S551A or S5549A/S551A point mutants failed to restore HPK1-mediated and TCR-mediated NF-κB activation and IL-2 expression in CARMA1-deficient T cells. Thus, we identify HPK1 as a kinase specific for CARMA1 and suggest HPK1-mediated phosphorylation of CARMA1 as an additional regulatory mechanism tuning the NF-κB response upon TCR stimulation.

Germinal center kinases in immune regulation

“…2,3 Intact holokinase MAP4K1 mediates T-cell receptor (TCR)-induced activation of nuclear factor of kappa light chain in B cells (NF-kB) through phosphorylation of the adaptor protein caspase recruitment domain containing protein 11 (CARMA1). 4 Upon TCR stimulation, SLP-76 interacts with and activates MAP4K3, which directly activate protein kinase C (PKC)-theta, leading to NF-kB activation. 5 MAP4K3-deficient mice showed resistance to experimental autoimmune encephalomyelitis.…”

NF-κB signaling pathways regulated by CARMA family of scaffold proteins

“…Indeed, PDK1, AKT, TAK1, IKKβ, CK1α, and HPK1 have been shown to associate with or may phosphorylate CARMA1 [67][68][69][70][71]. Moreover, some kinases may contribute to CARMA1 phosphorylation outside the linker region.…”

Down-regulation of B Cell Receptor Signaling by Hematopoietic Progenitor Kinase 1 (HPK1)-mediated Phosphorylation and Ubiquitination of Activated B Cell Linker Protein (BLNK)

“…The role of HPK1 in NF-B activation is complex. HPK1 binds to and phosphorylates CARMA1, resulting in the activation of IKK␤/NF-B (15,16). In addition, HPK1 can be processed by caspases into the N-terminal kinase domain and C-terminal regulatory domain in apoptotic cells (17); the cleaved HPK1 C-terminal fragment has a suppressive activity on NF-B activation (18).…”