title: Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder.
abstract: Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.
authors: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan.
authors: Mari
authors: M
authors: Sekiguchi
authors: Syouichi
authors: S
authors: Katayama
authors: Naoya
authors: N
authors: Hatano
authors: Yasushi
authors: Y
authors: Shigeri
authors: Noriyuki
authors: N
authors: Sueyoshi
authors: Isamu
authors: I
authors: Kameshita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D007525:Isoelectric Focusing; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015518:Rett Syndrome / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D014020:Tissue Extracts; C075637:amphiphysin; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C533690:CDKL5 protein, mouse