Cyclin-dependent kinase-like 5 (CDKL5/STK9) is a poorly understood protein kinase mutated in CDKL5 disorder. This disease is characterized by seizure onset before 3 months of age, severely impaired gross motor, language and hand skills and subtle but shared physical characteristics. It is not clear why mutations in CDKL5 cause this disease, especially as the cellular processes controlled by CDKL5 remain unknown and the cellular targets of CDKL5 and the mechanisms controlling its activity are also unclear.

A team of researchers in the John Rouse lab led by Ivan Munoz and Michael Morgan (with help from Florian Weiland and Mateusz Gregorczyk), in collaboration with Julien Peltier and Matthias Trost now based at University of Newcastle, has found the first cellular substrates of the CDKL5. Quantitative phospho-proteomic screening identified MAP1S, CEP131 and DLG5 as physiological substrates of CDKL5, and these were confirmed as CDKL5 substrates in a series of follow up experiments. All three substrates are all involved in control of the cytoskeleton, suggesting that CDKL5 may regulate cytoskeletal function. This idea is consistent with recent data implicating CDKL5 in the control of primary cilia.

The identification of CDKL5 substrates enabled the team to answer some important questions about CDKL5. Firstly, the phosphorylated serine in all three substrates lies in the motif RPXSA. Experiments with synthetic peptides revealed that CDKL5 can phosphorylate Ser residues that lie in the motif RPX[S/T][A/G/P], although it is possible that residues other than A/G/P can be tolerated C-terminal to the phospho-acceptor serine. Although CDKL5 is present in cytoplasm and nucleus, no nuclear substrates of CDKL5 were identified; it will take more work to find these substrates but a prediction is that they may be phosphorylated on the motif RPX[S/T][A/G/P]. Secondly, the CDKL5 team was able to test the functional consequences of the pathological CDKL5 mutations for the first time. These experiments revealed that CDKL5 mutations which cause CDKL5 disorder caused a severe reduction in the ability to phosphorylate MAP1S and CEP131 in cells. Thirdly, the CDKL5 team showed that the activity of CDKL5 is controlled by autophosphorylation of a key tyrosine residue in the kinase T-loop. These results were published in the EMBO Journal.

There are several exciting applications made possible by the identification of the first cellular substrates of CDKL5. Firstly, phospho-MAP1S and phospho-CEP131 antibodies can be used as biomarkers of CDKL5 activity. This will enable experiments to screen for stimuli that activate CDKL5, for example, and will allow the effectiveness of new treatments for CDKL5 disorder to be tested - gene replacement therapy and drugs that promote reading through nonsense mutations, for example. A major limitation, however, is that better antibodies will be required to allow analysis of the phosphorylation of endogenous MAP1S and CEP131. Recent identification of the Rab proteins as targets of the Parkinson’s disease kinase LRRK2 by the Alessi lab required the development of rabbit monoclonal antibodies to allow analysis of endogenous Rab phosphorylation in biological samples. The CDKL5 team demonstrated for the first time that pathogenic mutations in CDKL5 are loss-of-function in that they inhibit phosphorylation of MAP1S and CEP131. Highly sensitive phospho-specific antibodies may allow screening for small molecules, or genes that when deleted by genome editing, rescue the phosphorylation of MAP1S and CEP131 phosphorylation in CDKL5-defective cells. Such an endeavour may pave the way for new therapies to treat CDKL5 disorder. The CDKL5 team was funded by the Loulou Foundation (London) and the Medical Research Council.