WASH has a critical role in NK cell cytotoxicity through Lck-mediated phosphorylation

Natural killer (NK) cells are important effector cells of the innate immune system to kill certain virus-infected and transformed cells. Wiskott–Aldrich Syndrome protein (WASP) and SCAR homolog (WASH) has been identified as a member of WASP family proteins implicated in regulating the cytoskeletal reorganization, yet little is known about its function in lymphocytes. Here we demonstrate that WASH is crucial for NK cell cytotoxicity. WASH was found to colocalize with lytic granules upon NK cell activation. Knockdown of WASH expression substantially inhibited polarization and release of lytic granules to the immune synapse, resulting in the impairment of NK cell cytotoxicity. More importantly, our data also define a previously unappreciated mechanism for WASH function, in which Src family kinase Lck can interact with WASH and induce WASH phosphorylation. Mutation of tyrosine residue Y141, identified here as the major site of WASH phosphorylation, partially blocked WASH tyrosine phosphorylation and NK cell cytotoxicity. Taken together, these observations suggest that WASH has a pivotal role for regulation of NK cell cytotoxicity through Lck-mediated Y141 tyrosine phosphorylation.Natural killer (NK) cells are the first defense line against viral infections and tumors.¹ NK cell-mediated lysis of target cells requires the formation of immunological synapse between NK cells and target cells and subsequent delivery of lytic granules containing perforin and granzymes.^{2, 3} The importance of the actin cytoskeleton in this process has been well documented.⁴ However, the precise mechanism of actin reorganization in NK cells remains to be elucidated.Wiskott–Aldrich syndrome protein (WASP) is the first identified member of an actin regulator family.⁵ WASP family proteins contain a C-terminal domain that binds to and activates the Arp2/3 complex for cytoskeleton remodeling.⁶ In the absence of WASP, cytotoxic activity of NK cells is defective owing to impaired immune synapse formation and perforin localization.⁷ It has also been shown that WASP may be important for integration of NK cell signaling, particularly for nuclear translocation of NFAT2 and NF-κB during the activating receptor NKp46-dependent activation.⁸WASP and SCAR homolog (WASH) has been discovered as a new WASP family member.⁹ Subsequent studies show that WASH interacts with multiple proteins, including FAM21, to form a large core complex and regulate actin dynamics.¹⁰ WASH localizes to sorting and recycling endosomes, where WASH complex activates Arp2/3-mediated actin polymerization and controls the production of transport intermediates from endosome.¹¹ Unlike other WASP family members, WASH has distinct N-terminal domains, termed WASH homology domain 1 (WHD1) and tubulin-binding region (TBR).¹² Moreover, WASH has been shown to regulate recycling of many surface receptors via endosomal trafficking in activated T cells.¹³In our previous works, we found embryonic lethality and extensive autophagy in WASH-deficient mice. WASH recruits RNF2 to ubiquitinate AMBRA1 and inhibits the ubiquitination of Beclin1, a well-known moderator in autophage.^{14, 15} Of interest, WASH is located in cell nucleus and participates in hematopoietic stem cell differentiation through recruiting NURF complex to c-Myc promoter.¹⁶ However, the role and mechanism of WASH in NK cell function remain poorly understood.In this study, we show that inhibition of WASH expression with RNA interference or an inducible gene targeting system severely impair NK cell cytotoxicity through blockade of lytic granule polarization. In addition, Src family kinase Lck can interact with and induce tyrosine phosphorylation of WASH protein in human NK cells. These analyses provide the cellular and molecular mechanisms involved in the regulation of WASH function during NK cell activation.