Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domai
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RESEARCH
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Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain Szabolcs Pesti1, Annamária Balázs1, Roopesh Udupa1, Beáta Szabó2, Anna Fekete2, Gábor Bőgel1 and László Buday1,2*
Abstract Background: Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. Results: 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. Keywords: EphB1 receptor tyrosine kinase, Nck, Caskin1, SH3 domain, CD spectrum
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
* Correspondence: [email protected] 1 From the Department of Medical Chemistry, Semmelweis University Medical School, Budapest 1094, Hungary 2 Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest 1113, Hungary
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 sy
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