Growth is an essential process of life. Unchecked cellular growth, however, is a hallmark of cancer. Therefore, the process of cell division is often a target of cancer therapeutics (Steinmetz and Prota, 2018; Wieczorek et al., 2016). The macromolecular machine responsible for accurately segregating chromosomes during eukaryotic cell division is the bipolar mitotic spindle, a structure composed of dynamic microtubules organized by a multitude of microtubule-associated proteins (MAPs) (Hornick et al., 2010; Barisic and Maiato, 2016). Doublecortin-like kinase 1 (DCLK1), formerly known as DCAMKL1 and KIAA0369, is one such MAP that is also upregulated in a range of cancers, such as pancreatic, breast, bladder, colorectal, gastric, and hepatocellular carcinoma (Burgess et al., 1999; Lin et al., 2000; Li and Bellows, 2013; Meng et al., 2013; Qu et al., 2015; Liu et al., 2016; Fan et al., 2017; Kadletz et al., 2017; Jiang et al., 2018; Zhang et al., 2017). As a consequence, many studies have focused on developing small-molecule inhibitors against DCLK1 kinase activity in an effort to control cancer growth (Westphalen et al., 2017; Weygant et al., 2014; Ferguson et al., 2020). However, it is currently unclear if DCLK1 kinase activity, microtubule-binding activity, or both are involved in the molecule’s functions during cell division. Therefore, a mechanistic understanding of DCLK1, both at the molecular and biological level, is currently lacking.

DCLK1 is a member of the doublecortin (DCX) superfamily, which also includes DCX, DCDC2, and retinitis pigmentosa 1 (RP1), all of which are implicated in human disease (Westphalen et al., 2017; Reiner et al., 2006; Sullivan et al., 1999; Meng et al., 2005; Gleeson et al., 1998; Francis et al., 1999). At its N-terminus, DCLK1 contains two tandem DCX domains (DC1 or N-DC: aa 54–152 and DC2 or C-DC: aa 180–263) (Figure 1A), which are highly conserved among other family members (Lin et al., 2000; Reiner et al., 2006; Sapir et al., 2000; Kim et al., 2003a; Taylor et al., 2000). DCLK1 and its paralog, DCX, were originally identified and characterized for their functions during neuronal development, including neurogenesis and neuronal migration (Burgess et al., 1999; Gleeson et al., 1998; Francis et al., 1999; Bai et al., 2003; Burgess and Reiner, 2000; Jean et al., 2012). Although the roles of DCLK1 and DCX in neurodevelopment have been phenotypically described in vivo, the molecular basis for these observations remains ill-defined. Prior studies have shown that DCLK1 and DCX may act as microtubule stabilizers, nucleators, and regulators of microtubule-based transport (Liu et al., 2012; Moores et al., 2004; Moores et al., 2006; Bechstedt and Brouhard, 2012; Bechstedt et al., 2014; Lipka et al., 2016; Monroy et al., 2020; Ettinger et al., 2016; Patel et al., 2016). Dissecting the mechanisms by which DCLK1 binds to the microtubule can therefore provide insights into the microtubule-binding behaviors of other DCX family members and how they may be subverted in disease.

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The C-terminal portion of DCLK1 contains a serine/threonine kinase domain and an unstructured C-terminal tail that shares sequence similarities with calcium/calmodulin-dependent protein kinase I (CaMKI) (Shang et al., 2003; Edelman et al., 2005). For both DCLK1 and CaMKI, removal of a distal C-terminal ‘tail’ region results in an increase in kinase activity (Patel et al., 2016; Shang et al., 2003; Edelman et al., 2005; Goldberg et al., 1996). This mode of regulation has been well-studied for CaMKI, whose C-terminal tail makes direct contact with the kinase domain, directly inhibiting its enzymatic activity (Goldberg et al., 1996). However, it is unclear if, or how, the C-terminal tail of DCLK1 regulates its kinase domain. In addition, the physiological significance of DCLK1 kinase activity is unknown, even though it is a target for the development of kinase inhibitors due to its prominent role in cancer (Westphalen et al., 2017; Weygant et al., 2014; Ferguson et al., 2020). Additional information on the functional role of the DCLK1 kinase domain and how it is controlled would therefore be valuable for understanding how drugs can effectively target DCLK1 for therapeutic purposes.

Here we present a detailed examination of the microtubule-binding properties of DCLK1 and how they are regulated by its kinase activity. We find that DCLK1 autophosphorylates one key residue (T688) within its C-terminal tail via an intramolecular mechanism to strongly modulate its microtubule-binding affinity. Removal of the C-terminal tail or mutation of T688 results in an increase in phosphorylation of residues within both the DC1 and the DC2 domains, which in turn decreases microtubule binding. Furthermore, we observed that mutating four key phosphosites within DC1 of DCLK1 rescues microtubule binding in the construct lacking the C-terminal tail. Overall, our data led to a model in which DCLK1 autophosphorylates its C-terminal tail to modulate the activity of its own kinase domain and, subsequently, the level of phosphorylation within its microtubule-binding domains. To our knowledge, this is the first example of a self-regulatory MAP that can tune its microtubule-binding properties based on autophosphorylation state. Our results uncover a novel intramolecular regulation of microtubule binding within a prominent family of MAPs and may have implications for DCLK1’s known roles in tumor development and cancer progression.

Previous results have suggested that phosphorylation of DCLK1 occurs in part via autophosphorylation (Patel et al., 2016; Shang et al., 2003). To determine if DCLK1 phosphorylation is mediated by an inter- or intramolecular mechanism, we utilized an established kinase-dead mutant of DCLK1 (D511N) (Patel et al., 2016; Patel et al., 2021) and an active wild-type (WT) DCLK1 enzyme, both purified from bacteria (Figure 1—figure supplement 1A and Figure 1—figure supplement 2A). We did not observe trans-phosphorylation of DCLK1-D511N upon incubation with DCLK1-WT, although DCLK1-WT efficiently autophosphorylated itself in this assay (Figure 1—figure supplement 2B). Thus, under the conditions in our experiments, DCLK1 phosphorylation occurs via an intramolecular mechanism.

Removal of the C-terminal region of DCLK1 that follows the kinase domain results in an increase in kinase activity (Shang et al., 2003). How this region regulates enzymatic activity and autophosphorylation of DCLK1 and how phosphorylation of the molecule affects its microtubule-binding properties are open questions. We first compared the mobility of full-length mouse DCLK1-WT (aa 1–740) and a truncated DCLK1 lacking the C-terminal tail (ΔC: aa 1–648) to full-length kinase-dead DCLK1-D511N on a Phos-tag gel, which enhances the separation of differentially phosphorylated proteins (Figure 1B and Figure 1—figure supplement 1A-B; Kinoshita et al., 2009). We found that bacterially expressed DCLK1-WT and DCLK1-ΔC proteins migrated more slowly into the Phos-tag gel, indicative of higher levels of phosphorylation, compared to the non-phosphorylated DCLK1-D511N (Figure 1B). Using total internal reflection fluorescence microscopy (TIRF-M), we imaged sfGFP-tagged WT, ΔC, and D511N proteins binding to taxol-stabilized microtubules (Figure 1C) at concentrations differing by eightfold. Strikingly, DCLK1-ΔC did not bind to microtubules at either concentration tested, in stark contrast to DCLK1-WT and DCLK1-D511N, which both robustly bound to microtubules (Figure 1C–D). Notably, DCLK1-D511N bound microtubules more robustly at lower concentrations than DCLK1-WT. This is consistent with the prior work showing that D511N robustly stimulates tubulin polymerization (Patel et al., 2016). These experiments suggest that the C-terminal region regulates DCLK1 autophosphorylation, which in turn directly modulates its microtubule-binding affinity.

To test this possibility, we sought to evaluate the microtubule-binding behaviors of dephosphorylated DCLK1-WT and DCLK1-ΔC. We incubated the proteins with the Mn²⁺-dependent protein phosphatase, lambda phosphatase (λPP), which strongly dephosphorylated DCLK1-WT and DCLK1-ΔC as evidenced by the marked shifts on a Phos-tag gel without phospho-intermediate bands, but had little effect on the migration of D511N (Figure 1E and Figure 1—figure supplement 2C). The similar migration of DCLK1-WT and DCLK1-ΔC in the absence of phosphatase, coupled with the relatively larger migration shift of DCLK1-ΔC after treatment, suggests that DCLK1-ΔC is hyperphosphorylated compared to the WT protein, in agreement with previous results (Shang et al., 2003). In addition, anion exchange chromatograms also revealed a greater shift in the elution volume between phosphorylated and non-phosphorylated DCLK1-ΔC compared to the shift observed for phosphorylated vs non-phosphorylated WT protein, consistent with DCLK1-ΔC having a higher negative charge due to being hyperphosphorylated (Figure 1—figure supplement 1C). Using TIRF-M, we found that λPP-treated DCLK1-WT and DCLK1-ΔC bound to microtubules similarly to DCLK1-D511N (Figure 1F–G), similar to prior results showing that phosphatase-treated DCLK1 robustly stimulates tubulin polymerization (Patel et al., 2016). This further suggests that autophosphorylation modulates the microtubule-binding affinity of DCLK1 and that hyperphosphorylation of DCLK1-ΔC largely abolishes microtubule binding.

The high phosphorylation levels observed for both DCLK1-WT and DCLK1-ΔC suggested that these proteins phosphorylate themselves during bacterial expression. To determine the contributions of the C-terminal tail to DCLK1 function, we used a previously defined strategy to control the levels of autophosphorylation during expression (Patel et al., 2016; Patel et al., 2021). We co-expressed DCLK1-WT and DCLK1-ΔC with λPP in bacteria, followed by the subsequent removal of λPP from the DCLK1 preps via affinity and ion exchange chromatography. We compared DCLK1 proteins prepared in the absence or presence of λPP on a Phos-tag gel and observed that λPP co-expression substantially reduced phosphorylation levels of both DCLK1-WT and DCLK1-ΔC (Figure 2A). For all subsequent experiments, all DCLK1 protein variants were co-expressed with λPP. Upon incubation of dephosphorylated DCLK1 proteins with adenosine triphosphate (ATP), both DCLK1-WT and DCLK1-ΔC exhibited an increase in phosphorylation, but DCLK1-ΔC appeared to be entirely phosphorylated by 30 min, whereas DCLK1-WT displayed a number of phosphorylated intermediates even at 60 min (Figure 2A–B; 93.7% of DCLK1-ΔC protein shifts to the uppermost band after a 30-min incubation with ATP compared with 41.8% of DCLK1-WT protein after a 60-min incubation with ATP). Using TIRF-M, we determined the microtubule-binding affinities for DCLK1-WT and DCLK1-ΔC in the absence and presence of ATP (Figure 2C–E). We found that, in the absence of ATP, both proteins exhibited relatively similar microtubule-binding affinities (Figure 2C–E). After a 30-min incubation with ATP, the microtubule-binding affinity of DCLK1-WT moderately weakened, as evidenced by an ~2.5-fold increase in K_D (Figure 2D). However, incubation with ATP resulted in a dramatic approximately forty-onefold decrease in microtubule affinity of DCLK1-ΔC (Figure 2E). Interestingly, in the presence of ATP, DCLK1-ΔC was still present at regions of microtubule curvature, consistent with prior results that doublecortin proteins have a higher affinity for these regions in vitro (Figure 1—figure supplement 2D; Bechstedt et al., 2014). We also analyzed the binding behaviors of WT and ΔC on non-stabilized guanosine diphosphate (GDP) microtubule lattices grown from GMPCPP seeds and observed a similar decrease in bound DCLK1-ΔC in the presence of ATP (Figure 2F–G). Finally, we performed a microtubule co-sedimentation assay with WT and ΔC and found that while similar amounts of protein pelleted with microtubules in the absence of ATP, significantly less ΔC co-pelleted with microtubules in the presence of ATP (Figure 1—figure supplement 2E). These results indicate that the loss of its regulatory C-terminal tail results in aberrant DCLK1 hyperphosphorylation, leading to a dramatic loss of microtubule-binding affinity. Thus, the kinase activity of DCLK1 directly controls its association with microtubules via intramolecular phosphorylation, which in turn is regulated by the C-terminus of the protein.

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In order to determine how phosphorylation regulates the microtubule-binding affinity of DCLK1-ΔC, we performed liquid chromatography with tandem mass spectrometry (LC-MS/MS) of phosphorylated DCLK1-WT and DCLK1-ΔC proteins. For each phosphorylated residue identified, we counted the total number of DCLK1-WT and DCLK1-ΔC peptides containing the phosphorylated residue, and then calculated the percent of those peptides whose spectra revealed phosphorylation at that residue. Within the microtubule-binding region of DCLK1 (aa 44–263), we found that nine sites were more frequently phosphorylated in DCLK1-WT samples and 17 sites were more frequently phosphorylated in DCLK1-ΔC samples (Figure 3A–B). Of the 17 phosphorylation sites in DCLK1-ΔC, five either directly contact tubulin or are adjacent to residues that directly contact tubulin within the lattice (Manka and Moores, 2019). The architecture of DCLK1 suggests that it likely has the flexibility to autophosphorylate its N-terminal half due to an intrinsically disordered region between the DC2 domain and the kinase domain (Figure 3C; aa 263–374). These results indicate that the loss of microtubule binding of DCLK1-ΔC is due to an increase in phosphorylation at multiple sites, as opposed to a single site whose phosphorylation status dictates microtubule binding.

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The decrease in microtubule-binding affinity we observed for the DCLK1-ΔC construct in the presence of ATP could be due to a general increase in phosphorylation throughout the entire microtubule-binding region (aa 46–263) or due to phosphorylation at specific sites within the microtubule-binding DC domains. In order to determine the relative contributions of phosphorylation within the DC1 and DC2 domains to the decrease in the microtubule-binding affinity of DCLK1-ΔC, we mutated four conserved residues that showed the highest increase in phosphorylation within the DC1 (C_4A-DC1: S77, S83, S96, T143) or the DC2 domain (C_4A-DC2: T189, S193, T218, S228) (Figure 4 and Figure 1—figure supplement 1A-B, Figure 3—figure supplement 1A-B; Manka and Moores, 2019). We reasoned that if phosphorylation of these residues is responsible for the decreased microtubule-binding affinity of DCLK1-ΔC, then mutating these residues to alanines, which cannot be phosphorylated, should rescue the microtubule-binding defect of this construct in the presence of ATP. If, however, phosphorylation of these residues is not responsible for the decreased microtubule-binding affinity, then there should be no difference in binding between the DCLK1-ΔC and ΔC_4A-DC1 or C_4A-DC2 regardless of the presence of ATP.

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Using TIRF-M, we imaged DCLK1-ΔC, ΔC_4A-DC1, and C_4A-DC2 binding to taxol-stabilized microtubules in the presence or absence of ATP (Figure 4B–D). In the absence of ATP, all three DCLK1 proteins bound robustly to microtubules at both 5 nM and 20 nM. For all proteins at 5 nM, in the presence of ATP, there was substantially less DCLK1 on the microtubule (Figure 4B–C), similar to our previous results (Figure 2); however, there was a small, but significant increase in the amount of ΔC_4A-DC1 bound to the microtubule (Figure 4B–C). At 20 nM, in the presence of ATP, we observed significantly more ΔC_4A-DC1 and ΔC_4A-DC2 on the microtubule compared with ΔC, but this result was far more striking for ΔC_4A-DC1, which exhibited an approximately eightfold higher fluorescence intensity on the microtubule than ΔC (Figure 4B,D). These results indicate that mutating the residues in DC1 that are abnormally phosphorylated in the ΔC construct rescued microtubule binding by preventing phosphorylation within this domain. Mutating the phosphosites within DC2 also rescued microtubule binding, but to a lesser extent than the DC1 mutations. Therefore, aberrant phosphorylation of these residues in DCLK1-ΔC could indeed be responsible for the dramatic attenuation of microtubule binding.

We next wanted to determine the mechanism by which the C-terminal region of DCLK1 prevents hyperphosphorylation of the DC domains. We examined autophosphorylated DCLK1-WT by LC-MS/MS and identified two threonine residues in the C-terminal region (T687 and T688) that were consistently phosphorylated (Figure 3—figure supplement 1C). In order to understand how the C-terminal tail contributes to autophosphorylation, we mutated T687 and T688 to alanines individually (Figure 5A). For all of the experiments with these mutants, we co-expressed DCLK1 proteins with λPP to obtain a dephosphorylated protein preparation. We first evaluated the ability of these mutants to autophosphorylate using the Phos-tag gel system (Figure 5B–C). While DCLK1-WT and T687A exhibited a moderate increase in phosphorylation after a 30-min incubation with ATP, T688A appeared to be entirely phosphorylated at this same time point (Figure 5B–C; 13.2, 62.7, and 96.0% of protein shifts to the most phosphorylated band after a 30-min incubation with ATP for WT, T687A, and T688A, respectively). Therefore, phosphorylation of T688 within the C-terminal domain may be critical for the regulation of the kinase activity of DCLK1. To further elucidate the consequences of abolishing these phosphorylation sites, we used TIRF-M to determine microtubule-binding affinities for the DCLK1 phosphomutants in the presence or absence of ATP (Figure 5D–F). In the absence of ATP, all DCLK1 proteins exhibited relatively similar microtubule-binding affinities based on the dissociation constants derived from fluorescent saturation curves (Figure 5D–F). After a 30 min incubation with ATP, the microtubule-binding affinities of DCLK1-WT and T687A were comparable, whereas T688A displayed a dramatic reduction in microtubule binding with an approximately fortyfold increase in K_D (Figure 5F). We also performed a microtubule co-sedimentation assay with WT and T688A and found that while similar amounts of protein pelleted with microtubules in the absence of ATP, significantly less T688A co-pelleted with microtubules in the presence of ATP (Figure 5G). These results indicate that DCLK1 likely autophosphorylates residues within its C-terminal region in order to control aberrant hyperphosphorylation within its microtubule-binding domain.

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There could be consequences of autophosphorylation for DCLK1 function other than microtubule binding. We therefore analyzed the effect of autophosphorylation on DCLK1 conformation and on DCLK1 sensitivity to calpain cleavage. First, we fractionated the following proteins by sucrose density centrifugation: normally expressed DCLK1-WT, DCLK1-WT co-expressed with λPP, normally expressed DCLK1-T688A, DCLK1-T688A co-expressed with λPP, and DCLK1-D511N (Figure 5—figure supplement 1A–B). We observed a similar profile for all proteins on a 3–9% step gradient (Figure 5—figure supplement 1A–B), indicating these proteins adopt similar gross conformations regardless of phosphorylation state. DCLK1 contains two PEST (proline/P, glutamic acid/E, serine/S, threonine/T) domains that are targeted by calpain for proteolytic cleavage (Figure 1A; Patel et al., 2016; Burgess and Reiner, 2001). Upon incubation of calpain with λPP-co-expressed DCLK1-WT or DCLK1-T688A that had been first incubated in the absence or presence of ATP, we detected similar cleavage products of DCLK1 for both proteins under both conditions (Figure 5—figure supplement 1C). Therefore, autophosphorylation does not appear to affect cleavage of purified DCLK1-WT by calpain under our conditions. These results support a regulatory role for autophosphorylation in dictating the microtubule-binding affinity of DCLK1, without affecting the overall, gross conformation of the molecule or its sensitivity to calpain cleavage.

Overall, our study elucidates a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. We have found that DCLK1 autophosphorylates within its C-terminal tail to prevent aberrant hyperphosphorylation within its microtubule-binding domain. To our knowledge, this is the first example of a MAP whose binding is controlled by autophosphorylation. Based on the relevancy of DCLK1 to the progression of a variety of cancer types, understanding DCLK1 autoregulation is critical in determining its biological function in healthy versus disease states.

Autophosphorylation control of microtubule-binding affinity is likely to be controlled by cellular context. DCX is tightly regulated at the transcriptional level to ensure proper temporal expression during neuronal development (Francis et al., 1999; Edelman et al., 2005; des Portes et al., 1998; Gleeson et al., 1999). In contrast, members of the DCLK family are expressed during embryonic, post-embryonic, and adult periods, and are also expressed in a range of tissues outside the brain (Reiner et al., 2006; Edelman et al., 2005). This lack of temporal restriction may, therefore, necessitate autoregulation, as well as other modes of control, for the DCLK family in a range of cellular activities. It is tempting to speculate that DCLK1 autophosphorylation acts as a temporal switch to control microtubule-binding affinity under specific situations, and this period of activity could be extended or shortened by modulation by phosphatases, other kinases, and proteases.

Although DCX and DCLKs have been found to nucleate, tip-track, and bundle microtubules, the physiological functions of these proteins in regulating microtubule growth and organization remain elusive (Moores et al., 2004; Moores et al., 2006; Bechstedt and Brouhard, 2012; Bechstedt et al., 2014; Ettinger et al., 2016). Understanding the conserved mechanisms by which these paralogs bind to microtubules is essential in ascribing molecular functions to DCX family members, most of which are implicated in disease (Reiner et al., 2006). Prior work has demonstrated that isolated DC domains cannot stimulate microtubule polymerization or effectively bind microtubules, indicating both domains are required in tandem (Sapir et al., 2000; Kim et al., 2003a; Taylor et al., 2000; Kim et al., 2003b). The contributions of the individual DC domains are still controversial. Initial cryo-electron microscopy (EM) structures of DCX on microtubules revealed only a single bound DC domain, which was hypothesized to be DC1 (Moores et al., 2006; Fourniol et al., 2013). However, a subsequent study showed that specifically blocking DC2, but not DC1, prevented DCX from interacting with microtubules, suggesting DC2 is critical for lattice binding (Burger et al., 2016). Recent cryo-EM data of DC1 and DC2 bound to microtubule lattices in different nucleotide states unveiled that DC2 binds to the guanosine triphosphate (GTP) microtubule lattice, while DC1 prefers the GDP microtubule lattice (Manka and Moores, 2019). This model is consistent with observations that DCX tracks the growing plus-end of the microtubule, which is specifically disrupted by missense mutations in DC2 (Bechstedt et al., 2014). Our data indicate that in the absence of the C-terminal tail (or mutation of T688), DCLK1 aberrantly phosphorylates itself within both the DC1 and DC2 domains, but it is primarily the phosphorylation within the DC1 that reduces the microtubule-binding affinity of the molecule. Mutating four phosphosites within DC1 restored microtubule binding for DCLK1-ΔC in the presence of ATP; however, we also observed a small, but significant, rescue of microtubule binding upon mutating four phosphosites within DC2, indicating the importance of this domain (Figure 4). It will be imperative in the future to determine the individual and tandem roles of the DC domains across the DCX superfamily in a range of microtubule processes.

We have found that phosphorylation at different sites within DCLK1 has distinct effects on the molecule. Phosphorylation within the C-terminal region is essential to restrict the kinase activity of DCLK1, preventing hyperphosphorylation within the DC2 domain and eradication of microtubule binding. This mechanism could prove important in directing the kinase activity of DCLK1 to orthogonal molecular substrates (Koizumi et al., 2017) instead of itself. Alternatively, the C-terminal tail of DCLK1 could be cleaved under specific cellular situations to release it from the microtubule (Burgess and Reiner, 2001; Sarkar et al., 2017). Our assays provide evidence that autophosphorylation does not appear to affect calpain cleavage or the overall gross conformation of DCLK1. However, these results do not rule out the effects of phosphorylation on processing by other proteolytic enzymes or potentially small or dynamic conformational changes that would not be apparent in our assays. Future studies will be necessary to expand upon these results both in vitro and in vivo and determine how DCLK1 function is regulated within the cell.

The region of DCLK1 spanning the kinase domain and C-terminal tail is 46% identical to the comparable region of CaMKI (Patel et al., 2016; Shang et al., 2003; Edelman et al., 2005). The flexible C-terminal tail of CaMKI serves as a regulatory switch; it forms multiple interactions with the kinase domain and keeps it in an inactive conformation (Goldberg et al., 1996). Our data implicating the C-terminal tail of DCLK1 in preventing kinase hyperactivity combined with prior structural data propose a similar model for DCLK1 (Patel et al., 2016). Furthermore, we have identified a residue (T688) within the DCLK1 C-terminal tail that is critical in modulating kinase activity. Whether the DCLK1 C-terminal tail interacts stably or dynamically with the kinase domain and when DCLK1 may need to switch from a microtubule-bound to -unbound state within the cell are open questions. Overall, the evidence for a conserved mechanism governing kinase activity for CaMKI and DCLK1 and how this could go awry in disease provide exciting new avenues for future exploration.

Finally, this study has implications for one of the greatest human adversaries: cancer. There are over 100 discrete forms of cancer, each with multiple causes (National Cancer Institute, 2020). Numerous studies have found that DCLK1 is upregulated and acts as an oncogene in a range of cancers including pancreatic, colorectal, gastric, bladder, and breast cancer (Li and Bellows, 2013; Meng et al., 2013; Qu et al., 2015; Liu et al., 2016; Fan et al., 2017; Kadletz et al., 2017; Jiang et al., 2018; Zhang et al., 2017). Due to the emerging body of evidence implicating DCLK1 in tumorigenesis, the protein appears to be a promising target for not just one, but for several types of cancers (Westphalen et al., 2017; Weygant et al., 2014; Ferguson et al., 2020). However, the complex intramolecular mechanism of DCLK1 must be thoroughly dissected before the field will be able to develop therapeutically effective drugs. For example, in light of our work, developing kinase inhibitors may not prove to be an effective means of controlling DCLK1’s microtubule-binding functions, because WT and kinase-dead DCLK1 bind with similar affinities to microtubules, and prior studies have shown that they stimulate tubulin polymerization to similar extents (Patel et al., 2016). Future studies on the biological functions of DCLK1 microtubule-binding and kinase activity during the initiation and progression of cancer cell proliferation and migration will provide fundamental insights into how DCLK1 contributes to this malady and how it can be adequately targeted.

All data generated or analyzed during this study are included in the manuscript or as source files.

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Author details

1. Regina L Agulto

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

2. Melissa M Rogers

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

3. Tracy C Tan

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Amrita Ramkumar

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Present address

   Synthego Corporation, Suite C, Redwood City, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Ashlyn M Downing

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Present address

   Ayni Therapeutics, Inc, Santa Cruz, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Hannah Bodin

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Julia Castro

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

8. Dan W Nowakowski

   N Molecular Systems, Inc, Palo Alto, United States

   Contribution

   Data curation

   Competing interests

   No competing interests declared

9. Kassandra M Ori-McKenney

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   kmorimckenney@ucdavis.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-2051-2495

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