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PI3K Links NKG2D Signaling to a CrkL Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion¹

Colin M. Segovis^*, Renee A. Schoon^*, Christopher J. Dick^*, Lucas P. Nacusi^*, Paul J. Leibson^* and Daniel D. Billadeau^2,*,

^* Department of Immunology and Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN 55905

The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the National Institutes of Health (Grants CA47752 and AI65474 to D.D.B.). D.D.B. is a Leukemia and Lymphoma Society Scholar.

² Address correspondence and reprint requests to Dr. Daniel D. Billadeau, Department of Immunology and Division of Oncology Research, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail address: billadeau.daniel{at}mayo.edu

³ Abbreviations used in this paper: CS, cytotoxic synapse; CMAC, 7-amino-4-chloromethylcoumarin; IP, immunoprecipitation; KIR, killer Ig-related receptor; MICA, MICA expressing BaF3 cells; MTOC, microtubule organizing center; SFDA, 5-sulfofluorescein diacetate; siCrkL, siRNA directed against CrkL; siNeg, negative control siRNA; siRNA, small interfering RNA; WR, Western Reserve.

⁴ The online version of this article contains supplemental material.