Strength and duration of TCR signaling are important in regulating T cell development and lineage commitment. As a key component of the TCR signaling pathway, Itk has been found to modulate several aspects of thymocyte maturation, including positive selection, negative selection, and conventional versus innate CD8+
T cell development (11
). These observations suggest that Itk activity is involved in fine-tuning TCR signaling, and that this, in turn, regulates T cell fate decisions. Further investigation of this process would be greatly aided by a mouse model in which Itk kinase activity is increased relative to wild-type Itk. Previous efforts to increase Itk activity by overexpression have uniformly been unsuccessful (36
). The data presented in this report provide a biochemical explanation for these failures. Quite unlike the Src family kinases, there are to date no known mutations that activate Itk signaling. To our knowledge, Itk(BtkSH3)
is the first Itk variant to show activity greater
than that of the wild type enzyme in T cells. Thus, in the future, a knock-in mouse expressing Itk(BtkSH3)
in place of wild-type Itk could provide a system for examining T cell development and lineage commitment in cells with increased Itk kinase activity.
The quantitative effects of modulating Itk self-association on T cell receptor signaling events are similar to those seen in studies of the Src family kinase, Lck. Src kinases, including Lck, are negatively-regulated by an intramolecular association between the SH2 domain and a phosphorylated tyrosine in the C terminal tail that is absent in the Tec kinases. A comprehensive examination of Lck autoinhibition was recently reported in which this intramolecular interaction is either disrupted or strengthened by sequence changes close to the phosphotyrosine (39
). Lck-deficient Jurkat T cells were reconstituted with wild-type Lck or Lck mutants, and T cell receptor signaling leading to Erk activation was examined. Similar to our findings for wild-type Itk and Itk(BtkSH3)
, modulation of the Lck autoinhibitory interaction led to detectable, but modest changes in Erk activation. Together these studies indicate that shifting the equilibrium between active and inactive kinase conformations alters the strength of TCR mediated signaling but does so within a limited range of outcomes. This is in contrast to mutations located in active sites that have pronounced, all or nothing, effects on downstream signaling.
In the current study, the sequence changes that we have introduced into full length Itk have been limited to the SH3/SH2 interface. As already mentioned, the isolated PH domain of Itk also forms intermolecular self-associated complexes (26
) and might therefore play a significant role in Itk clustering in T cells. Itk membrane association occurs via PH domain interactions with phosphatidylinositol (3,4,5) trisphosphate in a T cell stimulation dependent fashion (40
). Localization of Itk at the membrane could favor PH/PH intermolecular interactions resulting in further stabilization of self-associated Itk complexes. Indeed, the contribution of the PH domain to Itk self–association may be minimal in systems such as the NIH 3T3 cells used here for co-immunoprecipitation experiments, but significantly more pronounced in the context of the T cell membrane environment. Hence, the modest functional effects observed upon altering the SH3/SH2 interface could be due to a dominant role for the PH domain that has not been affected in the Itk(BtkSH3)
mutant. When more detailed structural insights into the PH/PH domain interface become available, this region of the Itk regulatory structure can be probed for specific sequence changes that disrupt PH domain self-association but are silent with respect to the membrane binding function of the PH domain. It seems likely that such mutations, by themselves or in combination with mutations in the SH3/SH2 interface, may shift the equilibrium further away from the self-associated form of Itk leading to further enhancement of Itk signaling in T cells.
Specific intermolecular self-association has been characterized for a number of protein systems (44
). Within the kinase superfamily this mechanism activates receptor kinases by promoting trans auto-phosphorylation both within and outside of the protein kinase domain (45
). In another example of activation by intermolecular association, the anti-viral protein kinase PKR dimerizes via phosphotyrosine-dependent binding to double-stranded RNA (46
). Alternatively, inhibition by dimerization occurs for the receptor-like protein tyrosine phosphatase-α where an inhibitory ‘wedge’ on one molecule inserts into the catalytic site of another molecule (48
). A crystal structure of the kinase domain from yeast Snf1 also reveals a dimeric arrangement that putatively impedes catalytic activity by steric means (49
). Likewise, a crystal structure of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reveals a regulatory segment that sterically blocks substrate binding to the catalytic site (50
). The CaMKII kinase domain itself is intrinsically active and dimer formation brings the regulatory segment into position to inhibit activity.
In light of these examples, a simple mechanistic model for Itk autoinhibition by self-association would invoke steric blockage of the catalytic site. However, it has been well documented that the isolated Itk kinase domain exhibits little or no catalytic activity (28
) suggesting the possibility of an alternative autoinhibition mechanism. We have previously reported a kinetic analysis of a series of Itk fragments and have shown that the SH2 domain and linker between SH2 and kinase domains are required to achieve wild-type levels of activity (28
). Like the Csk kinase (51
), the SH2 and SH2-kinase linker region make direct contact with the kinase domain to stabilize the active conformation of Itk. Thus, inhibition of Itk catalytic activity by SH3/SH2 mediated self-association could be explained if the intermolecular interactions between Itk regulatory domains (particularly those involving the SH2 domain) compete with activating interactions between the SH2 and kinase domains.
T cell activation itself produces signals that compete with the self-association equilibrium and would shift the population of Itk in the cell toward a monomeric state. Specifically, exogenous binding partners such as transiently produced phospholigands would compete directly with Itk self-association by interfering with the SH2/SH3 interaction interface (9
). If self-association is disfavored, the regulatory domains could then adopt the catalytically competent conformation (28
). Conversely, in the absence of such activating factors, Itk might remain self-associated and autoinhibited.
It is also possible to envision a role for Itk autoinhibition following activation of Itk by TCR engagement; in this case, self-association would be one mechanism that could terminate Itk signaling (in addition to the activity of phosphatases). In binding studies of the Itk SH3 and SH2 domains we found that Y180 phosphorylation in the SH3 domain enhances affinity for the Itk SH2 domain (54
). Together with the observations that autophosphorylation at Y180 in the SH3 domain has no effect on Itk kinase activity and occurs in cis (54
) it is reasonable to suggest that autophosphorylation may be a first step toward turning off Itk-mediated signaling, by promoting intermolecular self-association leading to a drop in kinase activity. Indeed, a previous report suggests that Itk clustering in T cells occurs following membrane association (27
While the precise mechanistic details of when and how intermolecular association of Itk modulates its activity remain a question, this mode of autoinhibition for a non-receptor tyrosine kinase suggests that reagents (or mutations) that shift the equilibrium toward the self-associated state could dampen T cell activation while disfavoring self-association will increase Itk activity and concomitant T cell activation.