title: Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5).
abstract: Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase known to be associated with X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amph1) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amph1 as a model substrate. Amph1 served as an efficient CDKL5 substrate, whereas Amph2, a structurally related homologue of Amph1, was not phosphorylated by CDKL5. The sequence around the Amph1 phosphorylation site is RPR(293)SPSQ, while the corresponding sequence in Amph2 is IPK(332)SPSQ. To define the amino acid sequence specificity of the substrate, various point mutants of Amph1 and Amph2 were prepared and phosphorylated by CDKL5. Both Amph2(I329R) and Amph1 served as efficient CDKL5 substrates, but Amph1(R290I) did not, indicating that the arginyl residue at the P -3 position is critical for substrate recognition. With regard to prolyl residues around the phosphorylation site of Amph1, Pro-291 at the P -2 position, but not Pro-294 at the P +1 position, is indispensable for phosphorylation by CDKL5. Phosphorylation experiments using various deletion mutants of Amph1 revealed that the proline-rich domain (PRD) (amino acids 247-315) alone was not phosphorylated by CDKL5. In contrast, Amph1(247-385), which comprised the PRD and CLAP domains, served as an efficient CDKL5 substrate. These results, taken together, suggest that both the phosphorylation site sequence (RPXSX) and the CLAP domain structure in Amph1 play crucial roles in recognition and phosphorylation by CDKL5.
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Syouichi
authors.initials: S
authors.lastname: Katayama
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D006801:Humans; D051379:Mice; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009419:Nerve Tissue Proteins; C075637:amphiphysin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D017346:Protein Serine-Threonine Kinases; C114482:CDKL5 protein, human; C533690:CDKL5 protein, mouse