Antigen-induced signaling through the TCR complex triggers the orchestrated, dynamic movement of signaling machinery to defined regions of the IS. However, the mechanisms that couple cellular localization to signaling activity for most pathway components remain poorly understood. In this report we identify the kinesin GAKIN as an inhibitory component of the TCR pathway that regulates the signaling output and localization of the multi-domain scaffold CARD11 to control the extent of TCR-mediated activation of NF-κB.
It has been previously established that after TCR engagement, CARD11 undergoes a signal-dependent transition from an inactive to an active signaling scaffold () that requires the PKCθ-mediated phosphorylation of the ID. ID phosphorylation causes it to disengage from intramolecular interactions with the CARD and Coiled coil domains and allows the recruitment of signaling proteins to CARD11 to induce IKK activation (). This transition in CARD11 likely takes place in the central portion of the IS where PKCθ resides during signaling and where the IKK complex is recruited in a CARD11-dependent manner (Hara et al., 2004
Schematic of GAKIN action during TCR signaling
Our data reveal a subsequent step in TCR signaling at which GAKIN acts to limit CARD11 activity (). During this step, GAKIN associates with CARD11 in a manner that can reduce Bcl10 binding and promote the redistribution of CARD11 away from the PKCθ-rich central portion of the synapse. The redistribution of CARD11 to distal regions of the APC:T cell contact limits the interval during which CARD11 and PKCθ co-occupy the center of the synapse, and likely determines the duration of active CARD11 signaling in that region.
Several mechanisms could explain how occupancy of CARD11 in the center of the synapse would determine CARD11 signaling activity. First, PKCθ must phosphorylate the ID on at least two serines to convert CARD11 into an active scaffold. It is possible that the dwell time of CARD11 in the central region of the contact determines the fraction of CARD11 molecules that become fully phosphorylated by PKCθ, especially if these phosphorylation events are reversible by the action of a phosphatase. Second, active signaling may be restricted to the center of the contact by the CARD11-independent PKCθ-mediated activation of other signaling components that must work in concert with CARD11 in the pathway. For example, Shambharkar et al
. demonstrated that during TCR signaling, IKK kinase activation requires both the CARD11-dependent ubiquitination of IKKγ and the CARD11-independent phosphorylation of IKKα/β by TAK1, which was shown to be activated and associate with PKCθ during TCR signaling in a CARD11-independent manner (Shambharkar et al., 2007
). The dwell time of CARD11 in the center of the synapse may thus determine the extent to which CARD11-dependent signaling events can couple to CARD11-independent steps.
It is possible that GAKIN transports CARD11 as cargo away from the center of the APC: T cell contact (), although our data does not address this directly. IS formation results in MTOC polarization to the contact (Smith-Garvin et al., 2009
), which orients microtubules such that they emanate away from the center of the IS with their plus ends distal to the MTOC. GAKIN is capable of plus-end directed motility on microtubules (Horiguchi et al., 2006
; Yamada et al., 2007
), and the orientation of microtubules at the IS could allow directional, GAKIN-mediated transport of CARD11 to a distal region. It is important to note that in T cells depleted of GAKIN by RNAi, CARD11 still redistributes to the distal region of the contact, albeit with altered kinetics. This delayed redistribution might be due to the action of the residual GAKIN in these cells, or to a distinct mechanism that functions with GAKIN in CARD11 redistribution.
Our data suggests several important roles for the motor domain of GAKIN during TCR signaling. The motor is required for the dynamic localization of GAKIN at the IS at early timepoints (), where CARD11-dependent signaling to the IKK complex likely occurs (Hara et al., 2004
). During signaling, the motor appears to override determinants in the other domains of GAKIN that appear to specify localization to the MTOC, because deletion of the motor results in confinement of GAKIN to the MTOC. In addition, the motor participates in an autoinhibitory intramolecular interaction with the MBS that can regulate motor activity (Yamada et al., 2007
) and that appears to regulate the GAKIN:CARD11 association since deletion of either of these domains enhances the ability of GAKIN to associate with CARD11ΔID (). Since GAKIN associates with CARD11 in a signal-dependent manner, it is likely that TCR signaling influences GAKIN conformation. It will be important to investigate this possibility, and the possibility that TCR signaling regulates a transition from an inactive to an active motor.
Interestingly, in overexpression studies that bypass the constraints imposed by signal-regulated cellular localization and protein conformation (), the motor itself is sufficient for GAKIN-mediated CARD11ΔID association and inhibition, but not necessary, since other GAKIN fragments including the MBS, TR1, and TR2, are also capable of associating with and inhibiting CARD11ΔID. We note that these studies do not address whether these domains bind CARD11 directly or indirectly since the association assays were done in the presence of other cellular proteins. Further studies will be required to understand how the multiple domains of GAKIN integrate function prior to and during antigen receptor signaling.
Bcl10 is an obligate component of the TCR pathway (Ruland et al., 2001
) and its recruitment to CARD11 is an essential step in TCR signaling (Blonska and Lin, 2009
). The dynamic association of GAKIN with CARD11 appears to reduce the fraction of CARD11 molecules that associate with Bcl10 during signaling (). Bcl10 is also targeted by other mechanisms implicated in the termination of TCR signaling to NF-κB, including its signal-induced phosphorylation and degradation (Lobry et al., 2007
; Scharschmidt et al., 2004
; Zeng et al., 2007
). The ability of GAKIN to compete with Bcl10 for CARD11 binding likely works in concert with these degradative mechanisms to accomplish the negative feedback that limits Bcl10 activity in TCR signaling.
The action of GAKIN on CARD11 activity represents a previously unrecognized mechanism used by lymphocytes to limit the extent of antigen receptor signaling to NF-κB. GAKIN is a signaling inhibitor that is poised to act after signaling has been initiated to tune signaling output. GAKIN does not act to terminate signaling, or to prevent signaling in the absence of receptor engagement. This is in contrast to other key inhibitory players in the pathway, including the E3 ligase Cbl-b, the deubiquitinases A20 and CYLD, and the kinase CK1α. Cbl-b appears to act upstream of CARD11 in the pathway, by inhibiting the extent of Akt and PKCθ activation following receptor engagement (Qiao et al., 2008
). Downstream of CARD11, A20 appears to dampen pathway output after signaling has begun by removing the K63-linked ubiquitin chains on MALT1 that contribute to the association between MALT1 and the IKK complex (Duwel et al., 2009
). CYLD has been shown to prevent spontaneous NF-κB activation in T cells in the absence of receptor engagement by inhibiting the ubiquitination and autoactivation of TAK1 (Reiley et al., 2007
). CK1α plays both positive and negative roles in this pathway, and appears to attenuate signaling by phosphorylating residues in the CARD11 ID, although the mechanistic outcome of these phosphorylation events remains unclear (Bidere et al., 2009
Multiple checkpoints appear to be independently required to tune the output of antigen receptor signaling, to prevent the unwarranted expansion and transformation of lymphocytes observed in lymphoma, and to ensure an appropriate adaptive immune response that does not harm the host. The role of GAKIN in TCR signaling may offer opportunities for the directed manipulation of lymphocyte activation and proliferation that might prove useful in the treatment of autoimmunity, immunodeficiencies, or lymphocytic cancers.