In this study, we demonstrated that three nPKC isoforms, PKC-ε, PKC-η, and PKC-θ, function in an ordered cascade to prime the IS for MTOC reorientation. These results establish a previously unappreciated role for nPKCs during IS formation, and provide insight into the dynamic regulation of cell polarity in lymphocytes.
The synaptic localization of PKC-θ and its crucial role in T cell development and signaling have been well established18, 19, 21
. Hence, it was not particularly surprising for us to find that PKC-θ was recruited to the plasma membrane downstream of TCR stimulation and that it was required for MTOC polarization. However, given previous work indicating that PKC-η is not recruited to the IS21
and that T cell activation is unaffected in
, it was surprising to find that both proteins did indeed accumulate synaptically, and that they were important for MTOC reorientation and downstream effector responses. Previous analyses of PKC-η recruitment were based largely on immunocytochemistry, and it is possible that the antibody used against PKC-η in these experiments was not effective for intracellular staining. Furthermore, the absence of a phenotype in PKC-ε-deficient T cells could simply reflect the redundancy between PKC-ε and PKC-η that we have documented in this study. In that regard, mice lacking both PKC-ε and PKC-η will be useful tools in future studies aimed at assessing the extent to which each protein can compensate for the other in various aspects of T cell function.
Using two-color TIRF imaging, we showed that PKC-ε and PKC-η are recruited to the region of TCR stimulation ~15 seconds before MTOC reorientation and 5-10 seconds before PKC-θ. We also found that PKC-ε and PKC-η accumulate in a broader plasma membrane zone than PKC-θ. This distinction was observed both on glass surfaces containing immobilized photoactivatable pMHC and on supported lipid bilayers containing mobile agonist pMHC and ICAM. Classical studies have divided the mature IS into three distinct supramolecular activation clusters (SMACs), a central SMAC (cSMAC) containing the TCR, a peripheral SMAC (pSMAC) enriched in integrins, and a distal SMAC (dSMAC) defined by the actin ring32
. Although there is some controversy over whether PKC-θ localizes to the cSMAC or the pSMAC22, 33
, it is generally accepted that its accumulation falls within the dSMAC. This hypothesis is consistent with our bilayer experiments, which showed that PKC-θ was contained by the actin ring at all timepoints. In contrast, PKC-ε and PKC-η were recruited evenly over the entire IS, overlapping extensively with the dSMAC as well as the more central synaptic domains. Taken together, our data indicate that PKC-ε and PKC-η are not constrained with PKC-θ to the center of the IS, and suggest that the determinants that guide their localization are at least partially distinct. It has been reported that signaling from the costimulatory receptor CD28 induces the formation of PKC-θ clusters in a ring around the cSMAC34
. Interestingly, although we did observe annular PKC-θ clustering in membranes containing pMHC, ICAM and the CD28 ligand CD80, PKC-ε and PKC-η did not form CD28-induced clusters of this kind (E. J. Q. and M. H., unpublished observations).
The spatiotemporal features of PKC-η and PKC-θ accumulation were largely recapitulated by constructs containing the respective tandem C1 domains of each protein, suggesting that the C1 region is primarily responsible for the observed differences in PKC-θ recruitment relative to PKC-ε and PKC-η. That PKC-ε and PKC-η accumulated faster and in a broader domain than PKC-θ suggests that they may have a higher affinity for DAG. However, it is also possible that PKC-ε and PKC-η, upon arrival at the IS, induce the production of an additional, non-DAG determinant that primes the IS for binding of the PKC-θ C1 region. This determinant could presumably even be non-lipid in nature, given that C1 domains from several PKC isoforms are known to engage in protein-protein interactions in the appropriate environments35
. In that regard, it is notable that we did not observe recruitment of PKC-δ to the plasma membrane in response to TCR stimulation, despite the fact that PKC-δ binds DAG in other contexts36
. Indeed, it is likely that DAG binding is only one of a number of features controlling C1 region-dependent nPKC localization in T cells.
The precise mechanisms by which PKC-θ, PKC-ε, and PKC-η promote MTOC reorientation remain to be determined. It is formally possible that PKC-θ alone couples plasma membrane signals to downstream cytoskeletal machinery, and that PKC-ε and PKC-η serve merely to recruit PKC-θ to the IS. We consider this model unlikely, however, in part because the subtle polarization phenotype of PKC-θ-deficient T cells indicates that other pathways can, in the right setting, partially compensate for PKC-θ deficiency. We favor an alternative model in which PKC-ε and PKC-η are coupled to the MTOC in a PKC-θ-independent manner for at least a portion of the response. Given the observed differences in recruitment behavior between PKC-θ, PKC-ε and PKC-η, it is tempting to speculate that PKC-ε and PKC-η are crucial for the initiation of polarization, while PKC-θ contributes during subsequent phases, possibly by refining the positioning of the MTOC at the IS. The identification of nPKC substrates and other associated proteins that function in this context will no doubt shed light on the precise roles played by each nPKC isoform. In that regard, it is intriguing to note that both PKC-ε and PKC-η associate with the formin mDia37
, which has been implicated in MTOC reorientation in T cells38
. It will be interesting to explore the possibility that mDia acts in concert with the nPKCs to modulate T cell polarity.
Using Marcksl1 as a probe of PKC function, we documented a rise in PKC activity at the IS that was coincident with the recruitment of PKC-ε and PKC-η. Marcksl1 overexpression also imposed a substantial delay in MTOC reorientation, consistent with the idea that PKC activity plays a central role in the process. This delay was quite intriguing to us because Marcksl1 has been shown to associate with dynein through the regulatory dynactin complex39, 40
, and therefore could conceivably play a role in dynein regulation during the polarization response. However, siRNA-mediated suppression of Marcksl1 had no effect on MTOC reorientation (data not shown), indicating Marcksl1 is probably not involved in this process at physiological concentrations.
PKCs are crucial for polarity induction in multiple cell types. However, in adherent cells such as fibroblasts and astrocytes, it is the aPKCs, rather than the nPKCs, that appear to play a central role11, 15
. aPKCs act together with components of the PAR (partitioning-defective) complex to establish polarity over a period of hours, which is substantially longer than the minutes required for MTOC reorientation in lymphocytes. Interestingly, aPKCs and PAR proteins have been observed to localize asymmetrically in T cell–APC conjugates3, 41, 42
, but only well after MTOC polarization to the IS has occurred. In addition, a recent report indicated that aPKC activity is required for sustained MTOC polarization (after 30 min) and the delivery of cytokines to the IS43
. These results, when taken together with our data, suggest that polarity establishment at the IS proceeds in two stages: a rapid, direction sensing phase mediated by lipid second messengers and nPKCs, followed by a consolidation phase that requires aPKCs and PAR proteins. One might imagine that the first stage would be crucial for fast events like cytotoxic killing, while the second stage would be necessary for facilitating slower processes, such as asymmetric cell division. Further identification and characterization of molecules required for the induction and/or the maintenance of lymphocyte polarity should enable us to assess more effectively the contribution of cell polarity to complex immune function.