Approximately 30% of proteins are synthesised and processed within the secretory pathway which comprises the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus and post-Golgi carriers. This includes proteins such as receptors and growth factors whose levels need to be tuneable to environmental conditions such as developmental stage, tissue damage, and nutrient availability. Whilst this can be controlled at a transcriptional level to some extent, the secretory machinery itself must also respond to cargo load and demand to regulate secretion as cell requirements change.
Despite the clear need to regulate protein flux through the secretory compartments, relatively little is understood about the signalling pathways acting on these organelles. The organelle most frequently studied in the context of signalling is the Golgi. For example, GPCR/PKA signalling has been shown to promote the budding of post-Golgi carriers from the trans-Golgi network1. Similarly, phosphoinositide signalling2 plays a role in Golgi exit, whilst Src signalling can regulate retrograde traffic back to the ER3. Numerous other pathways affect trafficking by altering Golgi structure, such as mitotic signalling cascades. However, with the exception of the unfolded protein response, little is known about signalling in the ER even though it is well placed as the first compartment in the secretory pathway to control proteostasis.
Recently a study was published showing Gα12 is activated by COPII component Sec24 at ER exit sites to promote anterograde transport in response to cargo load4. Now, in this new preprint by Centonze, the first ER-localised receptor tyrosine kinase (RTK) has been identified – Leukocyte tyrosine kinase receptor (LTK). Numerous components of the trafficking machinery at the ER are known to be phosphorylated, including multiple members of the COPII coat. Thus, the discovery of a receptor kinase at the ER which can regulate the abundance of ER exit sites (ERES) is a big step towards understanding the role of local (and potentially druggable) ER signalling in regulating protein flux.
Key points
In this preprint the authors produce several lines of evidence to demonstrate that LTK is an ER resident enzyme. Firstly, they show that LTK, but not the related protein ALK, colocalises with the ER marker CLIMP63 and also to some extent with the ERES marker Sec31. Secondly, they subject LTK to glycosidase treatments and find that it is sensitive to EndoH treatment targeting ER-localised core sugar modifications but not to PNGase treatment targeting glycan modifications made in the Golgi. Finally, the authors use the RUSH trafficking system to hook LTK in the ER before biotin-induced bulk release to show that LTK does not leave the ER whereas ALK does.
In the second part of the study the authors investigate the impact of LTK activity on the secretory pathway. Importantly, they find that knock down of LTK or inhibitor treatment reduces ERES number by 30-40% and hence retards anterograde trafficking to the Golgi. Consistent with this, the LTK interactome was found to include a large number of early secretory pathway proteins including Sec12, a GEF for Sar1 at ERES. Sec12 is predicted to have two tyrosine phosphorylation sites at residues 10 and 177 and the authors found that Sec12 phosphorylation is reduced in response to LTK inhibitor treatment. More specifically, Y10 but not Y177 is phosphorylated by LTK. Expression of a Sec12-Y10F mutant reduced the number of ERGIC53 punctae in cells much like LTK inhibition reduced ERES. Together this implies Y10 phosphorylation by LTK is important for ERES formation or maintenance, most likely through its effects on the Sar1 GTPase cycle since LTK inhibition also reduces Sar1 mobility and recruitment.
The future
I chose this preprint because of the wide-reaching implications of discovering an RTK at the ER. Not least is the fact that RTKs are druggable and so LTK may prove an important target for manipulation of secretory flux, especially as it appears to act globally on ERES numbers. The identification of Sec12 as an LTK target regulating ERES is certainly an interesting example of the importance of this discovery and the interactome presented in this preprint reveals the influence of LTK activity is likely to extend further. In general, RTKs sit as a centre point for sometimes quite expansive signalling networks with numerous downstream targets. Unpicking these pathways for LTK will no doubt hold a number of interesting surprises. Surprisingly, some of the proteins identified in the interactome are not commonly associated with the ER, for example ARL1 which resides at the TGN. Although the authors do not detect any LTK outside of the ER network, it may be possible that small amounts are cycling. Perhaps re-engineering of the RUSH system to use a Golgi localised hook may help in trapping LTK in sufficient amounts to see it. The big question remaining of course though is what ligands activate LTK to initiate signalling in the first place.
Questions for authors
What activates LTK?
By expansion, can the new list of LTK interactors help us to predict under what circumstances this pathway is likely to be used?
Some of the identified interactors are not thought of as ER localised and so I wonder how the authors might resolve this?
References
Muniz, M., Martin, M. E., Hidalgo, J.and Velasco, A. (1997). Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network. Proc. Natl. Acad. Sci. USA 94, 14461-14466
Di Paolo, G., De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature443, 651–657
Bard, F., Mazelin, L., Pechoux-Longin, C., Malhotra, V., and Jurdic, P. (2003). Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum. J. Biological Chemistry. 278, 46601-46606.
Subramanian, A., Capalbo, A., Ravi Iyengar, N., Rizzo, R., di Campli, A., Di Martino, R., Lo Monte, M., Beccari, A. R., Yerudkar, A., del Vecchio, C., Glielmo, L., Turacchio, T., Pirozzi, M., Geon Kim, S., Henklein, P., Cancino, J., Parashuraman, S., Diviani, D., Fanelli, F., Sallese, M., and Luini, A., (2019). Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum. Cell. 176, 1461–1476
Response: This is an excellent question. Others have suggested that the secreted proteins called FAM150A&B act as LTK ligands. However, this was mainly shown in zebrafish, which does not have an LTK version as mammals have it. We emphasise in our work that mammalian LTK is different from that of all other species. In addition, because LTK never leaves the ER, it is unlikely that it is activated by extracellular ligands. We are currently working to classify this controversy. In light of the fact that we found at least 5 cargo receptors to interact with LTK, we think that cargo-loaded cargo receptors act as triggers for LTK activation
2) By expansion, can the new list of interactors help us to predict under what circumstances this pathway is likely to be used?
Response: We think that LTK responds to the load of folded proteins and thereby regulates the capacity of the ER export to unload this organelle.
3) Some of the identified interactors are not thought of as ER localised and so I wonder how the authors might resolve this?
Response: We did indeed detect potential LTK interaction partners that localise to the Golgi/TGN. However, we have not validated these. In our work we show that LTK is resident in the ER. We cloned it into a RUSH construct that allows detection of synchronous traffic from the ER. No matter how long we wait, we never saw LTK leaving the ER. We can only speculate what these non-ER interactors might be: (i) unspecific interactors, (ii) maybe small amounts of LTK leave the ER that are beyond our detection limit (iii) the interaction with TGN proteins could be via ER-TGN contact sites. We and other might explore these exciting possibilities in the future
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