Activation of Tec family kinases in T cells occurs predominantly downstream of the TCR, and results in the regulation of various cellular processes such as lymphoid development, T cell activation and optimal T cell effector functions [1
]. Itk is the principal Tec kinase in T cells, and its activation is necessary to ensure proper immune responses [1
]. Nevertheless, many of the pathways connecting Itk-mediated signaling to immune-specific gene expression are not fully understood. The transcription factor TFII-I is a target of Btk signaling in B cells [8
], and thus we questioned whether TFII-I might also serve as a downstream target of Itk signaling in T lymphocytes. We focused our efforts into determining first, if TFII-I could be activated upon TCR and co-receptor signaling in T cells; and second, into uncovering some of the molecular determinants of Itk-TFII-I signaling in T cells.
Here, we provide evidence that TFII-I is tyrosine phosphorylated within 2’ of TCR and/or CD43 crosslinking in human T cells, as well as post-CD3/CD28 ligation in murine T lymphocytes and Jurkat cells ( and ). Strong and sustained TFII-I tyrosine phosphorylation was observed when both TCR and CD43, or TCR and CD28 were simultaneously engaged, relative to TCR, CD43, or CD28 ligation alone. These findings are in agreement with recent data showing that relative to single crosslinking, simultaneous crosslinking of CD43 and TCR [31
], or CD28 and TCR receptors [47
], resulted in stronger and prolonged ERK phosphorylation, enhanced Zap70 and ζ-chain phosphorylation, accompanied by high levels of IL-2 production and cell proliferation in human PBL-Ts. Consistent with this work, our current results show that the intensity of TFII-I tyrosine phosphorylation varied according to the extracellular stimulus involved; while CD43 ligation induced a short-lived and weaker TFII-I activation response relative to TCR ligation, simultaneous TCR and CD43, or TCR and CD28 crosslinking, resulted in a synergized activation response. Our data thus support the notion that TFII-I activation can be modulated in a trigger-specific manner to accommodate a cell’s environmental and functional requirements. The significance of stimulus-specific TFII-I activation remains to be determined; indeed, it will be interesting to explore which downstream signaling targets/genes are differentially induced following trigger-specific TFII-I activation. One might also investigate whether crosslinking of other co-stimulatory molecules such as CD2, and/or CTLA-4 augment TFII-I-dependent phosphorylation.
We also conclude that TFII-I activation follows a similar and rapid phosphorylation pattern in T and B cells (within 2’ of receptor crosslinking) (). These results are consistent with previous findings showing tyrosine phosphorylation of TFII-I in the Ramos B cell line as early as 2’ post-IgM crosslinking [8
]. When examining the kinetics of TFII-I tyrosine phosphorylation, we noticed that the response in Jurkat cells was prolonged past 15’ post-stimulation, whereas in B cells it appeared curtailed (declining at 10’ post-stimulation). The precise functional implications of these findings remain to be elucidated.
As a step towards uncovering whether Itk and TFII-I pathways could be linked in T cells, here we demonstrate both ex vivo
and in vitro
that TFII-I physically interacts with Itk in a constitutive manner (, , and S1
). This concurs with previous findings establishing that Btk-TFII-I interactions are also constitutive in murine and human B cells [9
]. However, our data indicate that in contrast with findings where Btk-TFII-I protein complexes dissociated upon 10’ IgM ligation in B cells [9
], Itk-TFII-I protein complexes are quite stable at different activation times in T cells ( and data not shown). These findings may represent a distinguishing feature between Btk-TFII-I and Itk-TFII-I signaling pathways in B and T cells. Interestingly, it has been demonstrated that while wild type Btk readily associates with TFII-I, a mutant version of Btk responsible for the Xid mutation, Btk-R28C, does not [9
]. Contrary to this, we have observed that the interaction of TFII-I with the homologous mutant Itk-R29C (Xid) is not abrogated ( and S3
). This could partly account for the intrinsic stability of Btk-TFII-I vs.
Itk-TFII-I protein complexes. Future experiments are thus required to fully resolve these differences and their functional outcomes.
We further establish that splice variants of TFII-I, (Δ, β, and α isoforms) are all capable of associating with Itk, in agreement with previous data demonstrating that these same isoforms interact with Btk [11
] (). Together, the data suggest that functional differences between Itk-TFII-I and Btk-TFII-I interactions are independent from the ability of Tec kinases to associate with distinct TFII-I isoforms.
Structural analysis of TFII-I and Itk interaction domains indicated that mutations in the proline rich, pleckstrin homology (Xid) or kinase domains of Itk did not abrogate TFII-I-Itk protein binding (). With regard to the TFII-I domains, we found that the first N-terminal 90 amino acids of TFII-I, although utilized, are not critical for Itk binding (). Regions required for Itk binding must reside beyond the first N-terminal 90 residues of TFII-I, and future work should target such regions. We have ensured in GST pull-down assays, comparable transfected protein levels of mutant and wild type TFII-I and Itk in whole cell extracts (Supporting Figure S3
). However, it is still possible that experimental conditions used in our pull-down assays may not allow to fully distinguish subtle differences in Itk binding with either full length TFII-I, or ΔN90 and N90 mutants. Thus, the involvement of the first 90 amino acids of TFII-I in binding Itk needs to be detailed more precisely. The resolution of our assays does not provide information of changes in secondary structure that could affect TFII-I binding to Itk. Consequently, future work should aim to expand the current findings. Notwithstanding, the first 90 amino acids of TFII-I are required for Btk binding, and thus, our findings represent an additional distinguishing feature between TFII-I-Btk and TFII-I-Itk protein interactions, whose functional significance remains to be determined.
Interestingly, preliminary data obtained with partial Itk constructs demonstrated that combined point mutations in the SH3 and kinase domains could impair Itk-TFII-I interactions (data not shown). A more comprehensive structure-function analysis of the Itk interaction domains (including the role of SH3, SH2, kinase and Tec regions) is therefore warranted. Taken together, our data provide new insights into the putative interaction domains between Itk and TFII-I.
Of relevance, our data show that TFII-I is tyrosine phosphorylated in the presence of wild type, but not kinase-dead Itk, suggesting that TFII-I is directly or indirectly, a downstream phosphorylation target of Itk (). It remains to be determined if Itk is capable of directly phosphorylating TFII-I. Importantly, mutations in both kinase and PH domains (Xid) of Itk severely impaired TFII-I tyrosine phosphorylation, suggesting that these regions are implicated in TFII-I tyrosine phosphorylation (). Moreover, decreased tyrosine phosphorylation of TFII-I in the presence of kinase-dead or kinase-dead/Xid mutants was not due to decreased association of TFII-I-Itk protein complexes. Our data also showed that mutations in the PR domain of Itk did not affect TFII-I-Itk interactions, but caused a decrease in TFII-I phosphorylation, although to a lesser degree than other Itk mutations. Future experiments are required to fully discern these implications.
It will also be significant to address if CD43 ligation activates Itk, or co-stimulates its activation in conjunction with anti-CD3. In agreement with these results, our in vitro
studies in Jurkat T cells, peripheral blood T lymphocytes and murine splenic T lymphocytes have consistently demonstrated TFII-I tyrosine phosphorylation upon extracellular stimulation. And, although the biochemical data suggest that Itk is capable of phosphorylating TFII-I, we do not exclude the possibility that TFII-I can be targeted by different kinases at distinct sites, simultaneously or not [11
]. Indeed, it has been documented that c-Src and Btk exert their effects on TFII-I through distinct and independent pathways, and moreover, that they target different tyrosine phosphorylation and interaction sites on TFII-I [11
]. Accordingly, other signaling pathways activating TFII-I might include Src-family kinases such as Lck and/or Fyn.
Presently, the functional consequences of TFII-I phosphorylation in T cells are not known. Given that TFII-I has multiple potential tyrosine phosphorylation sites, it is possible that these are utilized differentially. Future studies might focus on the determination of specific Itk-dependent TFII-I phosphorylation sites, and on the examination of putative phosphorylation differences in TFII-I in normal vs. Itk-deficient T cells. Experiments analyzing the phosphorylation status of TFII-I in T lymphocytes derived from Itk and/or Itk/Rlk knockout mice could shed some light into the role of Itk-dependent TFII-I activation.
As an additional approach to obtain a better assessment of the functional role of Itk-TFII-I signaling in T cells, we demonstrate in reporter assays that co-expression of Itk potentiates the TFII-I-driven transcriptional activity of the model c-fos
promoter, under basal and stimulating conditions (). These results are in concordance with previous work demonstrating that Btk is a potent inducer of TFII-I-mediated transcription [11
], suggesting a certain degree of similarity in the mechanisms governing TFII-I activation and gene regulation in various cell types. Because c-fos
transcription was potentiated when both Itk and TFII-I were co-expressed in fibroblasts, our results further suggest additive cooperation between TFII-I and Itk proteins in transcriptional regulation, presumably through protein-protein interactions localizing to the c-fos
promoter region. A comparison of Itk-TFII-I protein complexes in nuclear vs
. cytoplasmic cell compartments might provide insight into the regulatory mechanisms of Itk-TFII-I interactions and gene regulation. While our results suggest that Itk regulates the transcriptional activity of TFII-I during T cell activation, the physiological target genes involved in this pathway remain to be elucidated.
To conclude, mechanistic and functional analyses from our current study demonstrate for the first time, activation of the multifunctional transcription factor TFII-I in response to receptor engagement in T cells and further implicate Itk in such activation. Our work thus provides an initial step in understanding the biological role of this pathway in T cell function and development. Future experiments should aim to expand the present findings, particularly in the context of T cell function, leading to a better understanding of the mechanisms underlying Itk-TFII-I protein interactions and their importance in T cell signaling and gene regulation.