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.

CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling

“…While the MH2 domain is highly conserved among all SMADs, the different structure of the MH1 domain of SMAD3, which does not contain the 30‐amino‐acid insert that is present in SMAD2 , could explain the specificity of the CDKL5‐dependent phosphorylation of SMAD3. Though recent observation has raised the possibility that the RPXSA motif might represent a consensus sequence for phosphorylation by CDKL5 , other studies have identified CDKL5 phosphorylation targets that do not contain this consensus motif , suggesting the presence of a different consensus sequence for CDKL5 phosphorylation or of a protein folding that creates a noncontiguous CDKL5 phosphorylation motif . SMAD3 seems to belong to this latter group because the MH1 domain does not present a RPXSA motif.…”

A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens

“…The former resembles the wellknown Plk1 kinase motif (52), whereas the latter is informative for proline-directed kinases (53). For B*07 ligands, the prevalent phosphorylation motif is (R/K)PXpS, which loosely confirms to the substrate motif of CDKL5 (54). Therefore, our data seem to predict that individuals expressing specific HLA allotypes may be more prone to present phosphorylated peptides following aberrant function of specific kinases, providing another reason for further research into personalized therapies.…”

Cyclin-Dependent Kinase-Like 5 (CDKL5): Possible Cellular Signalling Targets and Involvement in CDKL5 Deficiency Disorder

“…prompted the researchers to consider it a proline-directed protein kinase [21]. However, using Amph1 mutants and catalytic fragment of CDKL5, we were able to determine that CDKL5 recognises the RPXS (A/P) sequence [78]. Furthermore, Muñoz et al have confirmed that full-length CDKL5 also recognises this motif using peptide substrates [70].…”

“…These observations suggest that CDKL5 recognises not only the amino acid sequence in the vicinity of the phosphorylation site but also some other external structural elements of the proteins to which it binds. By conducting a detailed analysis of the recognition mechanism using Amph1 as a model substrate, we found that the catalytic fragment of CDKL5 recognises a characteristic structural element of Amph1 known as CLAP [78]. Ser/Thr kinases are known to phosphorylate both Ser and Thr, but we have shown that CDKL5 preferentially phosphorylates the Ser residue of the substrate [78].…”

“…However, whereas these protein kinases efficiently phosphorylate substrates widely used to assess kinase activity, such as myelin basic protein and histones, CDKL5 does not appreciably phosphorylate such substrates. Furthermore, a catalytic fragment of CDKL5 does not phosphorylate metabotropic glutamate receptor 5 or septin 4, both of which contain an RPXSP sequence [78]. These observations suggest that CDKL5 recognises not only the amino acid sequence in the vicinity of the phosphorylation site but also some other external structural elements of the proteins to which it binds.…”