The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

The microtubule-associated protein (MAP), doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for the development of kinase inhibitors. However, the physiological roles of its kinase activity and how DCLK1 kinase activity is regulated remain elusive. Here we employ in vitro reconstitution with purified proteins to analyze the role of DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a single residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation largely within the doublecortin 2 (DC2) domain, which dramatically reduces the microtubule affinity of DCLK1. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule's ability to associate with microtubules. Overall, our results suggest a mechanism by which DCLK1 modulates its own kinase activity to tune its microtubule binding affinity, providing molecular insights into a unique form of autoregulatory control over microtubule binding activity within the broader family of MAPs. These results provide useful molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression. Author ContributionsM.R., A.R., and K.M.O.M. conceived of the project and designed the experiments. M.R., A.R., A.D., J.C. and H.B. cloned all DCLK1 constructs and purified the recombinant proteins. M.R. performed the in vitro TIRF-M experiments, the kinase assays, the sucrose gradients, the cleavage assays, and analyzed all of data. A.R. performed the ATPγS kinase assays. D.W.N. created the molecular models. M.R. and K.M.O.M. wrote the manuscript with input from all authors.

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