The biology of NK cells includes many processes that involve receptor-mediated signaling and actin assembly. Here we have demonstrated involvement of HS1, a protein uniquely poised at the interface between signaling and actin assembly, in transferring information between these two networks. First, we found that HS1 was necessary for many of the diverse processes essential for NK cell function, including adhesion, integrin activation, chemotaxis, lytic synapse assembly and cytolysis. Tyrosine phosphorylation of HS1 was necessary in every case, but we found that the two tyrosine residues of HS1 had distinct and separable functions. Second, we found that the initiation and/or maintenance of many of the receptor-based signaling pathways in NK cells, including those emanating from integrins and NK receptors, depend on one HS1 tyrosine residue. The integrin signaling function of HS1 was specific for LFA-1, the integrin required for the adhesion of NK cells to target cells, and only the most immediate signaling from NK receptors and integrins was independent of HS1. Therefore, HS1 occupies a central position in generating and maintaining signals, as well as directing actin assembly. Other pathways can promote or maintain actin assembly, which may reinforce or enhance the action of HS1, providing feedback to generate and maintain the signals ‘downstream’ of HS1.
One of our main goals was to determine the functional importance of tyrosine phosphorylation of HS1 in NK cells. Tyrosine phosphorylation is necessary for the function of cortactin in certain cell systems39,40
, and tyrosine phosphorylation of HS1 at Tyr378 and Try397 has been suggested to be important for activation induced by immune receptors, including B cell and T cell receptors and FcgRIII, and for platelet activation1,24–28
. Here we found that tyrosine phosphorylation of HS1 was also necessary for all of the functions of HS1 in NK cells. More notably, we found evidence that the two tyrosine residues of HS1 had distinct and separable functions. Tyr397 but not Tyr378 was required for NK cell–target cell synapse formation and cytolysis, as well as for NK receptor signaling. In addition, Tyr397 was specifically required for adhesion, cell spreading and nonchemotactic migration in response to LFA-1-based signals but not for the same processes mediated by VLA-4. In contrast, Tyr378 but not Tyr397 was necessary for NK cell chemotaxis.
We found that HS1 was necessary for NK cell signaling ‘downstream’ of immediate membrane-proximal events. Integrin ligation and activation initiates signaling that promotes actin cytoskeletal reorganization in NK cells4,5,16,17
. In terms of signaling from LFA-1, phosphorylation of Syk was unaffected by the loss of HS1, but activation of Lyn, Pyk2, Vav1, PI(3)K and Rho-family proteins was impaired. As for the activation of NK receptors, recruitment of the adaptor DAP10 did not depend on HS1 but the activation and recruitment of components ‘downstream’ of DAP10 did. In all these cases, the Y397 HS1 mutant but not the Y378 HS1 mutant was crucial for HS1 function.
HS1 is necessary for integrin activation. LFA-1 on the surfaces of circulating NK cells is maintained in a low-affinity state4,5
. Circulating NK cells can be activated by chemokines, which causes surface LFA-1 to shift to a high-affinity state as part of ‘inside-out’ integrin activation7
. Here we found that activation of LFA-1 on NK cells treated with the chemoattractant SDF-1a depended on HS1 Tyr397 independently of the total amount of LFA-1 on the cell surface. In contrast, physical ‘outside-in’ activation of LFA-1 by Mn2+
was unaffected. LFA-1 activation in lymphocytes requires activation of the GTPase Rap1 (refs. 8,9
) and the function of cytohesin-1, a multidomain adaptor11
. Here activation of Rap1 and the association of cytohesin-1 with LFA-1 were inhibited in cells expressing the Y397F HS1 mutant. Thus, HS1 Tyr397 seems to be ‘upstream’ of Rap1 and cytohesin-1 in the pathway to activating LFA-1. Rho-family GTPases are important for actin cytoskeleton dynamics ‘downstream’ of integrins32,41,42
. Vav1 is important for lymphocyte activation43–45
. Vav1 can interact with HS1, and this interaction depends on HS1 Tyr397 (ref. 1
). Here we found that in cells expressing the Y397F HS1 mutant and responding to LFA-1 engagement, Vav1 was not activated. In those cells, Vav1 did not associate with LFA-1 or localize to the lytic synapse. In contrast, engagement of VLA-4 by fibronectin did cause Vav1 activation in HS1-knockdown cells and cells expressing the Y397F HS1 mutant (data not shown).
We found that HS1 Tyr378 was specifically required for NK cell chemotaxis. A series of time-course assays designed to test the ability of NK cells to migrate toward target cells or chemoattractant showed that Tyr378 was critical for directed migration. Tyr378 became phosphorylated after the addition of chemoattractant and was required for the propagation of signaling induced by the chemoattractant. In cells expressing the Y397F HS1 mutant, initial activation of phospholipase C-b by chemoattractant was intact, but ‘downstream’ activation of PAK1, PI(3)K and Vav1 was impaired.
The responses of migrating leukocytes, including NK cells, to chemotactic gradients involves signaling through G protein-coupled receptors to Rho-family GTPases37,38
. Rac1 activity regulates activity at the front of a migrating cell, whereas Rho functions at the back. The integration of these signaling pathways determines the directional response to chemotactic signals41
. Here we found that chemoattractant stimulation activated Rac1 and Cdc42 and suppressed RhoA activity in NK cells. This activation of Rac1 and Cdc42 required HS1 Tyr378 but the downregulation of RhoA did not. Poor activation of Vav1 may have been the cause of the defective activation of Rac1 and Cdc42 induced by chemoattractant and integrin signaling. Thus, the Rho-family G proteins and Vav1 are involved ‘downstream’ of both Tyr378 and Tyr397 in response to chemokine and integrin stimulation, respectively.
We have established that HS1 and, more notably, phosphorylated regulation of HS1 are prominent in specific NK functions, including adhesion. We have determined that phosphorylation of HS1 Tyr397 was critical for integrin-mediated signaling required for adhesion to ICAM-1 in the context of substrate or adhesion to potential target cells. Alternatively, phosphorylation of HS1 Tyr378 was required for chemotaxis, which is critical for reaching potential target cells. As loss of HS1 does not entirely disrupt immediate-early integrin or NK receptor signaling, we feel that HS1 is an important ‘hub’ molecule poised between early signaling events and actin cytoskeletal remodeling required for many distinct NK functions.