Transforming growth factor β (TGF-β) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activation
Authors: William F Hawse
Mijoon Lee
William C Boggess
Richard T Cattley
Journal: Journal of Biological Chemistry (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: C-terminal Src kinase (CSK); T cell; T-cell biology; T-cell receptor (TCR); computer modeling
Affiliations: Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA., Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA., Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA .

Abstract:  The cytokine content in tissue microenvironments shapes the functional capacity of a T cell. This capacity depends on the integration of extracellular signaling through multiple receptors, including the T-cell receptor (TCR), co-receptors, and cytokine receptors. Transforming growth factor β (TGF-β) signals through its cognate receptor, TGFβR, to SMAD family member proteins and contributes to the generation of a transcriptional program that promotes regulatory T-cell differentiation. In addition to transcription, here we identified specific signaling networks that are regulated by TGFβR. Using an array of biochemical approaches, including immunoblotting, kinase assays, immunoprecipitation, and flow cytometry, we found that TGFβR signaling promotes the formation of a SMAD3/4-protein kinase A (PKA) complex that activates C-terminal Src kinase (CSK) and thereby down-regulates kinases involved in proximal TCR activation. Additionally, TGFβR signaling potentiated CSK phosphorylation of the P85 subunit in the P85-P110 phosphoinositide 3-kinase (PI3K) heterodimer, which reduced PI3K activity and down-regulated the activation of proteins that require phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) for their activation. Moreover, TGFβR-mediated disruption of the P85-P110 interaction enabled P85 binding to a lipid phosphatase, phosphatase and tensin homolog (PTEN), aiding in the maintenance of PTEN abundance and thereby promoting elevated PtdIns(4,5)P2 levels in response to TGFβR signaling. Taken together, these results highlight that TGF-β influences the trajectory of early T-cell activation by altering PI3K activity and PtdIns levels. 

Retracted: No

PubPeer comments:
 Dear authors, We have with great interest and excitement discussed your paper u201cTransforming growth factor u03b2 (TGF-u03b2) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activationu201d during our weekly literature meeting. Your paper convincingly shows the important role of TGFu03b2 receptor signaling in T cell activation, demonstrating an alternative role to transcriptional regulation. We greatly appreciated the well-designed experiments providing evidence for the underlying mechanisms downstream of TGFu03b2 receptor signaling as well as the simulations run to further support your data on downstream signaling events. Lastly, we strongly support your message on the great need for more work (or even a standardized pipeline) on T cell co-receptor signaling networks generated by various stimuli. Majority of T cell related papers focus only on T cell receptor stimulation and forget that the antigen recognition is supported modulated by a myriad of cues. However, after reading your paper, we were left wondering about a few things that we think would be interesting to see explored or commented, either here, or in future publications: 1. It was unclear if the murine CD4+ T cells activated through TCR and CD28 stimulation were nau00efve T cells or T cell blasts. In the methods section, you describe that following negative isolation: T cells were rested for 1h followed by CD3, CD28 and IL2 activation. However, is this to generate T cell blasts (if so, for how many days) or was this for short term stimulation of 10min? If so, the presence of IL2 may have been important to mention in the results section as well. Could TGFu03b2 receptor signaling play differential roles in nau00efve vs T cell blasts? 2. Were the concentrations of TGFu03b2 used for co-stimulations physiologically representative? 3. In fig 1D, you are reporting changes in FAK pY397 level upon TGFu03b2 stimulation, however you are not coming back to it later. Do you have a hypothesis regarding the role of FAK in the TGFu03b2 pathway? Where would you place FAK in your signaling model (presented in fig 7A)? 4. Have you considered to search your mass spectrometry results for phosphotyrosylated peptides? In our experience it is worth a try and sometimes gives unexpected hits (see PID: 25028575 for method) 5. In fig 3, phosphorylation of p85 Y458 is monitored and showed to be regulated by TGFu03b2 in a dose-dependent manner. However, we were unable to find Y458 listed on Phosphosite.org. The antibody you have used (CST, E3U1H) is said to be specific to both P85 pY458 and P55 pY199. The latter one constitutes of a motif DRLYEEY, which is the same as P85u03b1 pY467 (and quite similar to P85u03b2 pY464). What is the explanation for the discrepancy in numbering? Did you check for phosphorylation of any other sites on P85? Are other phosphosites on P85 similarly regulated by TGFu03b2 receptor signaling? We would greatly appreciate it if you could share your thoughts on our comments above, as this may aid us (and other readers) in better understanding your work and thus help us all contribute to a fruitful scientific discussion. , 
 #1 Hi Hanna, Hope you are well. Thank you very much for discussing our paper in your literature meeting and thoughtful comments. Iu2019ll do my best to briefly answer your questions, but if you would like a more detailed answer or to talk more about signaling, please e-mail me and we can set up a time to talk via zoom. 1. The T cells were nau00efve. They were not preactivated and only stimulated for 10 minutes as described in the figure legend. We included IL-2 so that our activation condition matches protocols used to generate Tregs in vitro culture. In pilot experiments where IL-2 was included or not included, we didnu2019t see much of a difference in the phosphorylation of some target proteins (eg ZAP70, LCK, AKT). The high-affinity component of the IL-2 receptor isnu2019t expressed this early, which could account for that. I would bet that IL-2 has different signaling impacts on nau00efve versus T cell blasts, but I donu2019t have data on that topic. 2. Thinking about the concentrations of cytokines used during in vitro polarization experiments versus in vivo settings is interesting. We selected concentrations of TGFB to be in the range used for Treg in vitro polarization experiments. My best guess is that T cells probably u201cseeu201d various concentrations of TGFB depending on the nature of the microenvironment and their location relative to cells generating TGFB. For most key signaling proteins we looked at in the paper, we did dose-response studies with varying doses of TGFB to partially address your question. We do indeed see that the activation status of many proteins is regulated as a function of TGFB concentration. 3. We are fascinated with FAK biochemistry and its function in T cells. Work from other groups and our own, demonstrate that PtdIns(4,5)P2 activates FAK autophosphorylation. In our model and data presented in the paper, TGFB signaling blocks PI3K activity resulting in a buildup of PtdIns(4,5)P2, which we believe drives FAK activation. Our previous publication entitled u201cT cells transduce T-cell receptor signal strength by generating different phosphatidylinositolsu201d in JBC demonstrated that PtdIns(4,5)P2 was required to activate FAK and that FAK was required for optimal FoxP3 expression. We have ongoing projects looking at FAK in T cell biology as we think there are still open questions as to its function in promoting Treg differentiation. 4. We did not process the data to look for phosphorylation. We now have pipelines set up to enrich phosphopeptides and have a paper under review using that technology. 5. We picked the numbering based on Cell Signaling Technologies product sheet for product # 17366 that listed the antibody as recognizing phosphorylated murine P85 on Tyr458. We are interested in PI3K assembly and are currently looking into PTMs that impact its activity and interaction network. I hope that your research is going well. Hope to keep in touch. All the best, Bill ,