Abstract
Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Huse, M., Quann, E.J. & Davis, M.M. Shouts, whispers, and the kiss of death: directional secretion in T cells. Nat. Immunol. 9, 1105–1111 (2008).
Stinchcombe, J.C. & Griffiths, G.M. Secretory mechanisms in cell-mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 23, 495–517 (2007).
Chang, J.T. et al. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315, 1687–1691 (2007).
Combs, J. et al. Recruitment of dynein to the Jurkat immunological synapse. Proc. Natl. Acad. Sci. USA 103, 14883–14888 (2006).
Martin-Cofreces, N.B. et al. MTOC translocation modulates IS formation and controls sustained T cell signaling. J. Cell Biol. 182, 951–962 (2008).
Quann, E.J., Merino, E., Furuta, T. & Huse, M. Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells. Nat. Immunol. 10, 627–635 (2009).
Kuhne, M.R. et al. Linker for activation of T cells, ζ-associated protein-70, and Src homology 2 domain-containing leukocyte protein-76 are required for TCR-induced microtubule-organizing center polarization. J. Immunol. 171, 860–866 (2003).
Lowin-Kropf, B., Shapiro, V.S. & Weiss, A. Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism. J. Cell Biol. 140, 861–871 (1998).
Martin-Cofreces, N.B. et al. Role of Fyn in the rearrangement of tubulin cytoskeleton induced through TCR. J. Immunol. 176, 4201–4207 (2006).
Newton, A.C. Protein kinase C: poised to signal. Am. J. Physiol. Endocrinol. Metab. 298, E395–E402 (2010).
Etienne-Manneville, S. & Hall, A. Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr. Opin. Cell Biol. 15, 67–72 (2003).
Fan, J. et al. PKCδ clustering at the leading edge and mediating growth factor-enhanced, but not ecm-initiated, dermal fibroblast migration. J. Invest. Dermatol. 126, 1233–1243 (2006).
Rosenberg, M. & Ravid, S. Protein kinase Cγ regulates myosin IIB phosphorylation, cellular localization, and filament assembly. Mol. Biol. Cell 17, 1364–1374 (2006).
Volkov, Y., Long, A., McGrath, S., Ni Eidhin, D. & Kelleher, D. Crucial importance of PKC-βI in LFA-1-mediated locomotion of activated T cells. Nat. Immunol. 2, 508–514 (2001).
Suzuki, A. & Ohno, S. The PAR-aPKC system: lessons in polarity. J. Cell Sci. 119, 979–987 (2006).
Ma, J.S., Haydar, T.F. & Radoja, S. Protein kinase Cδ localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity. J. Immunol. 181, 4716–4722 (2008).
Ma, J.S. et al. Protein kinase Cδ regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL. J. Immunol. 178, 7814–7821 (2007).
Altman, A. & Villalba, M. Protein kinase C-θ (PKCθ): it's all about location, location, location. Immunol. Rev. 192, 53–63 (2003).
Manicassamy, S., Gupta, S. & Sun, Z. Selective function of PKC-θ in T cells. Cell. Mol. Immunol. 3, 263–270 (2006).
Letschka, T. et al. PKC-θ selectively controls the adhesion-stimulating molecule Rap1. Blood 112, 4617–4627 (2008).
Monks, C.R., Kupfer, H., Tamir, I., Barlow, A. & Kupfer, A. Selective modulation of protein kinase C-θ during T-cell activation. Nature 385, 83–86 (1997).
Sims, T.N. et al. Opposing effects of PKCθ and WASp on symmetry breaking and relocation of the immunological synapse. Cell 129, 773–785 (2007).
Gruber, T., Thuille, N., Hermann-Kleiter, N., Leitges, M. & Baier, G. Protein kinase Cɛ is dispensable for TCR/CD3-signaling. Mol. Immunol. 42, 305–310 (2005).
Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007).
Petersen, N.O., Hoddelius, P.L., Wiseman, P.W., Seger, O. & Magnusson, K.E. Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. Biophys. J. 65, 1135–1146 (1993).
Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).
Bunnell, S.C., Kapoor, V., Trible, R.P., Zhang, W. & Samelson, L.E. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14, 315–329 (2001).
Arbuzova, A., Schmitz, A.A. & Vergeres, G. Cross-talk unfolded: MARCKS proteins. Biochem. J. 362, 1–12 (2002).
Higginbotham, H., Bielas, S., Tanaka, T. & Gleeson, J.G. Transgenic mouse line with green-fluorescent protein-labeled Centrin 2 allows visualization of the centrosome in living cells. Transgenic Res. 13, 155–164 (2004).
Sun, Z. et al. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature 404, 402–407 (2000).
Marsland, B.J., Soos, T.J., Spath, G., Littman, D.R. & Kopf, M. Protein kinase Cθ is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses. J. Exp. Med. 200, 181–189 (2004).
Dustin, M.L. Hunter to gatherer and back: immunological synapses and kinapses as variations on the theme of amoeboid locomotion. Annu. Rev. Cell Dev. Biol. 24, 577–596 (2008).
Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).
Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C θ translocation. Immunity 29, 589–601 (2008).
Colon-Gonzalez, F. & Kazanietz, M.G. C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim. Biophys. Acta 1761, 827–837 (2006).
Stahelin, R.V. et al. Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cδ. J. Biol. Chem. 279, 29501–29512 (2004).
Eng, C.H., Huckaba, T.M. & Gundersen, G.G. The formin mDia regulates GSK3β through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts. Mol. Biol. Cell 17, 5004–5016 (2006).
Gomez, T.S. et al. Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse. Immunity 26, 177–190 (2007).
Jin, T., Yue, L. & Li, J. In vivo interaction between dynamitin and MacMARCKS detected by the fluorescent resonance energy transfer method. J. Biol. Chem. 276, 12879–12884 (2001).
Yue, L., Lu, S., Garces, J., Jin, T. & Li, J. Protein kinase C-regulated dynamitin-macrophage-enriched myristoylated alanine-rich C kinase substrate interaction is involved in macrophage cell spreading. J. Biol. Chem. 275, 23948–23956 (2000).
Ludford-Menting, M.J. et al. A network of PDZ-containing proteins regulates T cell polarity and morphology during migration and immunological synapse formation. Immunity 22, 737–748 (2005).
Oliaro, J. et al. Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms. J. Immunol. 185, 367–375 (2010).
Bertrand, F. et al. Activation of the ancestral polarity regulator protein kinase Cζ at the immunological synapse drives polarization of Th cell secretory machinery toward APCs. J. Immunol. 185, 2887–2894 (2010).
Abeyweera, T.P., Merino, E. & Huse, M. Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells. J. Cell Biol. 192, 675–690 (2011).
Acknowledgements
We thank D. Littman (New York University) for PKC-θ-deficient mice; W. Marks and the Memorial Sloan-Kettering Cancer Center Mouse Genetics Core Facility for assistance with generating the C1-GFP mice; R. Wedlich-Soldner (International Max Planck Research School) for Lifeact-RFP; A. Hall (Memorial Sloan-Kettering Cancer Center), S. Rotenberg (Queens College) and M.O. Li (Memorial Sloan-Kettering Cancer Center) for reagents and advice; E. Alonzo for assistance with functional studies; N. Bantilan for technical support; S.S. Yi and the Memorial Sloan-Kettering Cancer Center Microchemistry Core Facility for peptide synthesis; A. Hall for critical reading of the manuscript; and members of the Huse and Li laboratories for helpful comments. Supported by the Searle Scholars Program (M.H.), the Cancer Research Institute (M.H.) and the US National Institutes of Health (Ruth L. Kirschstein National Research Service Award to E.J.Q., R01-AI087644 to M.H. and R01-AI083408 to G.A.-B.).
Author information
Authors and Affiliations
Contributions
E.J.Q. and M.H. designed the experiments; E.J.Q. collected and analyzed the data; X.L. did the Marcksl1 experiments; G.A.-B. assisted with MATLAB programming and data analysis; and M.H. wrote the manuscript (with help from E.J.Q. and G.A.-B.).
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 (PDF 2492 kb)
Supplementary Video 1
PKC-θ accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 2666 kb)
Supplementary Video 2
PKC-ɛ accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 2515 kb)
Supplementary Video 3
PKC-η accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 1649 kb)
Supplementary Video 4
PKC-η and PKC-θ display distinct spatiotemporal patterns of recruitment. (MOV 5617 kb)
Supplementary Video 5
PKC-ɛ and PKC-θ display similar spatiotemporal patterns of recruitment. (MOV 2539 kb)
Supplementary Video 6
Marcksl1 is depleted from the region of TCR stimulation prior to MTOC reorientation. (MOV 3146 kb)
Rights and permissions
About this article
Cite this article
Quann, E., Liu, X., Altan-Bonnet, G. et al. A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells. Nat Immunol 12, 647–654 (2011). https://doi.org/10.1038/ni.2033
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.2033
This article is cited by
-
Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization
Communications Biology (2022)
-
Recent insights of T cell receptor-mediated signaling pathways for T cell activation and development
Experimental & Molecular Medicine (2020)
-
miR-34a as hub of T cell regulation networks
Journal for ImmunoTherapy of Cancer (2019)
-
Origins of the cytolytic synapse
Nature Reviews Immunology (2016)
-
Regulation of T cell signalling by membrane lipids
Nature Reviews Immunology (2016)