title: Dok-1 tyrosine residues at 336 and 340 are essential for the negative regulation of Ras-Erk signalling, but dispensable for rasGAP-binding.
abstract: Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling.
authors.affiliation: Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Shinohara
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Yasuda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yamanashi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010649:Phenylalanine / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction; D014170:Transformation, Genetic; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000037:antagonists & inhibitors*
chemical_list: D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D010649:Phenylalanine; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; D048049:Extracellular Signal-Regulated MAP Kinases