Two Dok Protein Crystal Structure Of The Ptb Domain And Its Associated Functions

dok proteins are substrates of tyrosine kinases. They play a role in the recruitment and assembly of specific signal transduction molecules as adaptor. They contain a pleckstrin homology (PH) domain and a phospho-tyrosine-binding (PTB) domain, which are thought to mediate the association with the plasma membrane and with phosphotyrosine-containing motifs, respectively. The carboxy-terminal part of doks contain multiple tyrosine phosphorylation sites and likely functions as a molecular platform for signal complex assembly induced by activated PTKs. How doks specifically recognize the receptor tyrosine kinases is the key for understanding their functions.We have isolated a full-length cDNA that encodes a 36kDa protein from fetal brain cDNA library and named this gene as dok5. Northern blot indicated that dok5 highly expressed in skeletal muscle and can be detected in brain and heart. It is different from its counterpart in mouse that specifically expressed in brain. So far, it has been known there are five members in the dok family, dok-1-3 are mainly expressed in hematopoietic tissues and inhibit mitogen-activated protein (MAP) kinase signaling, cell proliferation, and cellular transformation, dok-4 is expressed in many tissues. dok5 is mainly expressed in skeletal muscle, it does not bind rasGAP, and plays a positive role in activation of the MAP kinase pathway.We cloned the dok1 and dok5 full-length cDNA and expressed their PTB domains, and then the recombinant proteins were purified by affinity, anion exchange and gel filtration chromatography. Crystals of dokl, dok5 and dokl's complex with the peptides deriving from Ret have been obtained by vapour-diffusion method. Biacore experiments showed dokl PTB domain can bind to the peptides deriving from Ret, but cannot bind to the peptides deriving from TrkA and IL-4R, although the Shc and Irs1 PTB domain can bind to them respectively. dok5 PTB domain cannot bind to all of menu The structures of them were solved by multiple anomalous diffraction method and the complex structure was determined by molecular replacement method. The structures of dokl and dok5's PTB domains both adopt the conserved pleckstrin homology domain fold, a seven-stranded, antiparallel P-sandwich that is capped at one end by a a-helix. In complex, the Ret phosphopeptide fills an L-shaped cleft on the domain. The C terminal residues of the peptide form a type I β turn. Some residues that conserved in doks and IRS' play an important role in recognizing of phosphotyrosine and some specifical residues in phosphopeptide motif. The substrate specificity of doks PTB domain may due to some specific residues in them. These studies have established the basis for further research.