Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function

Reviewer #1 (Public Review):

The Eph receptor tyrosine kinase family plays a critical function in multiple physiological and pathophysiological processes. Hence, understating the regulation of these receptors is a highly important question. Through extensive experiments in cell lines and cultured neurons, Chang et.al show that the signaling hub protein, MYCBP2 positively regulates the overall stability of a specific member of the family, EPHB2, and by that the cellular response to ephrinBs. Overall, this work sheds light on the divergence in the regulatory mechanisms of the Eph receptors family. The physiological importance of this new regular mechanism awaits discovery.

Reviewer #2 (Public Review):

Members of the EphB family of tyrosine kinase receptors are involved in a multitude of diverse cellular functions, ranging from the control of axon growth to angiogenesis and synaptic plasticity. In order to provide these diverse functions, it is expected that these receptors interact in a cell-type-specific manner with a diverse variety of downstream signalling molecules.

The authors have used proteomics approaches to characterise some of these molecules in further detail. This molecule, myc-binding protein 2 (MYCBP2) also known as highwire, has been identified in the context of establishment of neural connectivity. Another molecule coming up on this screen was identified as FBXO45.

The authors use classical methods of co-IP to show a kinase-independent binding of MYCBP2 to EphB2. They further showed that FBXO45 within a ternary complex increased the stability of the EphB2/MYCBP2 complex.

To define the interacting domains, they used clearly designed swapping experiments to show that the extracellular and transmembrane domains are necessary and sufficient for the formation of the ternary complex.

Using a cellular contraction assay, the authors showed the necessity of MYCBP2 in mediating the cytoskeletal response of EphB2 forward signalling. Furthermore, they used the technically challenging stripe assay of alternating lanes of ephrinB-Fc and Fc to show that also in this migration-based essay MYCBP2 is required for EphB mediated differential migration pattern.

MYCBP2 in addition is necessary to stabilize EphB2, that is in the absence of MYCBP2, EphB2 is degraded in the lysosomal pathway.

Interestingly, the third protein in this complex, Fbxo45, was further characterized by overexpression of the domain of MYCBP2, known to interact with Fbxo45. Here the authors showed that this approach led to the disruption of the EphB2 / MYCBP2 complex, and also abolished the ephrinB-mediated activation of EphB2 receptors and their differential outgrowth on ephrinB2-Fc / Fc stripes.

Finally, the authors demonstrated an in vivo function of this complex using another model system, C elegans where they were able to show a genetic interaction.

Data shows in a nice set of experiments a novel level of EphB2 forward signalling where a ternary complex of this receptor with multifunctional MYCBP2 and Fbxo45 controls the activity of EphB2, allowing a further complex regulation of this important receptor. Additionally, the authors challenge pre-existing concepts of the function of MYCBP2 which might open up novel ways to think about this protein.

Of interest is this work also in terms of the development of the retinotectal projection in zebrafish where MYCBP2/highwire plays a crucial role, and thus might lead to a better understanding of patterning along the DV axis, for which it is known that EphB family members are crucial.

Overall, the experiments are classical experiments of co-immunoprecipitations, swapping experiments, collapse assays, and stripe assays which all are well carried out and are convincing.

Reviewer #3 (Public Review):

In this manuscript, Chang et al set out to find direct interactions with the Eph-B2 receptor, as our knowledge of its function/regulation is still incomplete. Using proteomic analysis of Hela cells expressing EPHB2, they identified MYCBP2 as a potential binder, which they then confirm using extensive biochemical analyses, an interaction that seems to be negatively affected by the binding of ephrin-B2 (but not B1). Furthermore, they find that FBXO45, a known MYCBP2 interaction, strongly facilitates its binding to EPHB2. Intriguingly, these interactions depend on the extracellular domains of EPHB2, something that is surprising given the fact that MYCBP2 is an intracellular protein. Finally, they find that, in contrast to what could be expected given the known function of MYCBP2 as a ubiquitin E3 ligase, it actually positively regulates EPHB2 protein stability, and function.

The strength of this manuscript is the extensive biochemical analysis of the EPHB2/MYCBP2/FBXO43 interactions. Most of the conclusions are warranted although I do not understand the physiological interpretation of how these proteins could interact in the extracellular space.

The attempt to extend the study to an in vivo animal using the worm is important. However, I find the results in the worm confusing and overly interpreted in their current form.