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- Open Access
- Published:
Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain
Cell Communication and Signalingvolume 10, Article number: 36 (2012)
Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.
Conclusion
Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.
Background
The Eph family is the largest family of receptor tyrosine kinases, at least, 14 different Eph kinases have been identified so far[1, 2]. These receptors can be divided into two subclasses (EphB and EphA) based on the cell surface ligand which they interact with. Ligands that interact with EphA receptors are generally attached to the cell surface via glycosylphosphatidylinositol (ephrinA ligands), while ligands that activate EphB receptors are transmembrane proteins (ephrinB ligands)[1, 2]. Eph receptor kinases and their ligands (ephrins) play a critical role in embryonic patterning, angiogenesis and neuronal targeting[2–4]. In the nervous system, EphB receptor kinases are enriched at excitatory synapses and are important during synapse and spine formation and maintenance[2].
Adaptor proteins, composed almost entirely of well-defined interaction domains, serve to link two functional members of a signaling pathway[5]. Nck family of adaptors comprising of SH2 and SH3 domains are implicated in the organization of actin cytoskeleton, cell movement, and axon guidance. Their SH2 domains bind specific phosphotyrosine residues on activated receptors or their substrates, whereas the SH3 domains bind proline-rich motifs on downstream target proteins[6]. It has been shown earlier that Nck associates with a number of Eph tyrosine kinases, including EphB1, EphB2, EphA2, EphA3 and EphA4[7–12]. For EphB1, the first tyrosine residue localized in the juxtamembrane region represents the binding site for the SH2 domain of Nck, while its SH3 domains may interact with paxillin, Nck-interacting kinase (NIK), or WASP[7–9, 13].
Neurons contain numerous scaffold proteins that are involved in the regulation of synaptic function[14]. Cask-interactive proteins (Caskins) also belong to this group of scaffold proteins[15]. Caskin1 and its isoform Caskin2 are multidomain proteins possessing six ankyrin repeats, a single SH3 domain, and two sterile α motifs (SAM domains) in the N-terminal part[15]. In contrast, there are no recognizable domains in the C-terminal part, which is dominated by a long proline-rich region[15]. Recently, we provided evidence that the entire proline-rich region of Caskin1 is intrinsically disordered. We also showed that the proline-rich region is biochemically functional, as it interacts with the adaptor protein Abl-interactor-2 (Abi2)[16]. In addition to CASK and Abi2, Caskin1 can also interact with Nck/Dock, neurexin2, synaptotagmin, and septin4[12, 16, 17]. A Caskin homolog has been recently discovered in Drosophila protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while the Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor kinase, adaptor, and scaffold proteins results in the phosphorylation of tyrosine residues 296 and 336 localized in the SH3 domain of Caskin1. Using CD spectroscopy we found that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.
Results
Nck interacts with Caskin1 in an SH3 domain-dependent manner
We and others have shown earlier that the scaffold protein Caskin1 interacts with Nck/Dock[8, 12, 16, 17]. To confirm and characterize the interaction between the two proteins, first,. As Figure1a shows Caskin1 interacts stably with Nck in vivo. To study the interaction in another system, V5 epitope-tagged Caskin1 and/or GFP-tagged Nck were transiently expressed in COS7 cells, and then cell lysates were immunoprecipitated with an anti-V5 antibody. When both proteins were coexpressed in cells, Caskin1 was capable of associating with Nck (Figure1b). Nck possesses an SH2 and three SH3 domains[6]. Although it has been shown earlier that the interaction between Caskin1 and Nck is mediated through SH3 domains[12], it has not been determined which SH3 domain(s) is responsible for the association. Therefore, to determine the required SH3 domains for the Caskin1/Nck interaction, full length Nck, the three SH3 domains together (Nck-SH3-all), and individual SH3 domains (SH3/1, SH3/2 and SH3/3) were generated as GST fusion proteins. These fusion proteins were immobilized on glutathione-agarose beads and used for protein precipitations from lysates of COS7 cells expressing V5-Caskin1. As seen in Figure1c, none of the individual SH3 domains of Nck was able to pull-down the scaffold protein. However, when all three SH3 domains were combined, either in the full length Nck or the Nck-SH3-all construct, Caskin1 was readily precipitated. These findings suggest that similar to many of the Nck-interacting proteins (described in the Discussion) Caskin1 requires all SH3 domains of Nck for high affinity interaction. Figure1d demonstrates that the majority of the Nck GST fusion proteins gave single bands after SDS-PAGE and Commassie-blue staining.
Nck interacts with Caskin1 through its SH3 domains. (a)..
Caskin1 is recruited to the EphB1 tyrosine kinase via Nck
It has been well documented that a number of tyrosine kinases or their substrates can recruit Nck through its SH2 domain[6]. One of the tyrosine kinase receptors present in the nervous system is EphB1 which was reported to bind to Nck through its phosphorylated Tyr-594 residue[7]. Therefore, we hypothesized that Nck could mediate an interaction between EphB1 and Caskin1. To address this question, first, HA-tagged EphB1 was transiently expressed in COS7 cells stimulated with ephrin B1 the ligand of the receptor tyrosine kinase. As Figure2a demonstrates expression of EphB1 alone in these cells induces activation of the kinase which is reflected in its tyrosine phosphorylation. Interestingly, addition of the ligand did not further increase the receptor tyrosine phosphorylation. Other groups also found that expression of EphB1 or EphA3 in cells leads to autoactivation of these receptor kinases[11, 19]. Next, we tested if Nck could bind to EphB1 in COS7 cells expressing the HA-tagged kinase. It was found that an antibody against the endogenous N