title: The Activating C-type Lectin-like Receptor NKp65 Signals through a Hemi-immunoreceptor Tyrosine-based Activation Motif (hemITAM) and Spleen Tyrosine Kinase (Syk).
abstract: NKp65 is an activating human C-type lectin-like receptor (CTLR) triggering cellular cytotoxicity and cytokine secretion upon high-affinity interaction with the cognate CTLR keratinocyte-associated C-type lectin (KACL) selectively expressed by human keratinocytes. Previously, we demonstrated that NKp65-mediated cellular cytotoxicity depends on tyrosine 7, located in a cytoplasmic sequence motif of NKp65 resembling a hemi-immunoreceptor tyrosine-based activation motif (hemITAM). HemITAMs have been reported for a few activating myeloid-specific CTLRs, including Dectin-1 and CLEC-2, and consist of a single tyrosine signaling unit preceded by a triacidic motif. Upon receptor engagement, the hemITAM undergoes phosphotyrosinylation and specifically recruits spleen tyrosine kinase (Syk), initiating cellular activation. In this study, we addressed the functionality of the putative hemITAM of NKp65. We show that NKp65 forms homodimers and is phosphorylated at the hemITAM-embedded tyrosine 7 upon engagement by antibodies or KACL homodimers. HemITAM phosphotyrosinylation initiates a signaling pathway involving and depending on Syk, leading to cellular activation and natural killer (NK) cell degranulation. However, although NKp65 utilizes Syk for NK cell activation, a physical association of Syk with the NKp65 hemITAM could not be detected, unlike shown previously for the hemITAM of myeloid CTLR. Failure of NKp65 to recruit Syk is not due to an alteration of the triacidic motif, which rather affects the efficiency of hemITAM phosphotyrosinylation. In summary, NKp65 utilizes a hemITAM-like motif for cellular activation that requires Syk, although Syk appears not to be recruited to NKp65.
authors.affiliation: Institute for Molecular Medicine, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Bauer
authors.affiliation: Institute for Molecular Medicine, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Wotapek
authors.affiliation: Institute for Molecular Medicine, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Zöller
authors.affiliation: Institute for Molecular Medicine, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Emilia
authors.initials: E
authors.lastname: Rutkowski
authors.affiliation: Institute for Molecular Medicine, Goethe University, 60590 Frankfurt am Main, Germany. Electronic address: alexander.steinle@kgu.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinle
mesh_terms: D015550:Cell Degranulation; D002460:Cell Line; D006801:Humans; D007113:Immunity, Innate; D061625:Immunoreceptor Tyrosine-Based Activation Motif; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology*; D055503:Protein Multimerization; D055630:Receptors, NK Cell Lectin-Like / Q000032:analysis / Q000276:immunology*; D000072377:Syk Kinase / Q000032:analysis / Q000276:immunology*
chemical_list: C548774:KLRF2 protein, human; D055630:Receptors, NK Cell Lectin-Like; D000072377:Syk Kinase
keywords: NKp65; cell surface receptor; hemITAM; innate immunity; lectin; natural killer cells (NK cells); signal transduction; spleen tyrosine kinase (Syk)