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.

Figure 1 with 2 supplements see all

Download asset Open asset

Figure 1—source data 1

Uncropped gels for the associated panels in Figure 1.

The red box indicates how the gel was cropped.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig1-data1-v2.pdf

Download elife-60126-fig1-data1-v2.pdf

Figure 1—source data 2

Uncropped gels.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig1-data2-v2.zip

Download elife-60126-fig1-data2-v2.zip

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.

Figure 2

Download asset Open asset

Figure 2—source data 1

Uncropped gel for the associated panel in Figure 2.

The red box indicates how the gel was cropped.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig2-data1-v2.pdf

Download elife-60126-fig2-data1-v2.pdf

Figure 2—source data 2

Uncropped gels.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig2-data2-v2.zip

Download elife-60126-fig2-data2-v2.zip

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.

Figure 3 with 1 supplement see all

Download asset Open asset

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.

Figure 4

Download asset Open asset

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.

Figure 5 with 1 supplement see all

Download asset Open asset

Figure 5—source data 1

Uncropped gels for the associated panels in Figure 5.

The red box indicates how the gel was cropped.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig5-data1-v2.pdf

Download elife-60126-fig5-data1-v2.pdf

Figure 5—source data 2

Uncropped gels.

https://cdn.elifesciences.org/articles/60126/elife-60126-fig5-data2-v2.zip

Download elife-60126-fig5-data2-v2.zip

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.

1. 1. Bai J
   2. Ramos RL
   3. Ackman JB
   4. Thomas AM
   5. Lee RV
   6. LoTurco JJ

   (2003) RNAi reveals doublecortin is required for radial migration in rat neocortex

   Nature Neuroscience 6:1277–1283.

   https://doi.org/10.1038/nn1153

   • PubMed
   • Google Scholar
2. 1. Barisic M
   2. Maiato H

   (2016) The tubulin code: a navigation system for chromosomes during mitosis

   Trends in Cell Biology 26:766–775.

   https://doi.org/10.1016/j.tcb.2016.06.001

   • PubMed
   • Google Scholar
3. 1. Bechstedt S
   2. Lu K
   3. Brouhard GJ

   (2014) Doublecortin recognizes the longitudinal curvature of the microtubule end and lattice

   Current Biology 24:2366–2375.

   https://doi.org/10.1016/j.cub.2014.08.039

   • PubMed
   • Google Scholar
4. 1. Bechstedt S
   2. Brouhard GJ

   (2012) Doublecortin recognizes the 13-protofilament microtubule cooperatively and tracks microtubule ends

   Developmental Cell 23:181–192.

   https://doi.org/10.1016/j.devcel.2012.05.006

   • PubMed
   • Google Scholar
5. 1. Burger D
   2. Stihle M
   3. Sharma A
   4. Di Lello P
   5. Benz J
   6. D'Arcy B
   7. Debulpaep M
   8. Fry D
   9. Huber W
   10. Kremer T
   11. Laeremans T
   12. Matile H
   13. Ross A
   14. Rufer AC
   15. Schoch G
   16. Steinmetz MO
   17. Steyaert J
   18. Rudolph MG
   19. Thoma R
   20. Ruf A

   (2016) Crystal structures of the human doublecortin C- and N-terminal domains in complex with specific antibodies

   Journal of Biological Chemistry 291:16292–16306.

   https://doi.org/10.1074/jbc.M116.726547

   • Google Scholar
6. 1. Burgess HA
   2. Martinez S
   3. Reiner O

   (1999) KIAA0369, doublecortin-like kinase, is expressed during brain development

   Journal of Neuroscience Research 58:567–575.

   https://doi.org/10.1002/(SICI)1097-4547(19991115)58:4<567::AID-JNR9>3.0.CO;2-T

   • PubMed
   • Google Scholar
7. 1. Burgess HA
   2. Reiner O

   (2000) Doublecortin-like Kinase Is Associated with Microtubules in Neuronal Growth Cones

   Molecular and Cellular Neuroscience 16:529–541.

   https://doi.org/10.1006/mcne.2000.0891

   • Google Scholar
8. 1. Burgess HA
   2. Reiner O

   (2001) Cleavage of Doublecortin-like kinase by calpain releases an active kinase fragment from a microtubule anchorage domain

   Journal of Biological Chemistry 276:36397–36403.

   https://doi.org/10.1074/jbc.M105153200

   • Google Scholar
9. 1. Castoldi M
   2. Popov AV

   (2003) Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer

   Protein Expression and Purification 32:83–88.

   https://doi.org/10.1016/S1046-5928(03)00218-3

   • Google Scholar
10. 1. des Portes V
    2. Pinard JM
    3. Billuart P
    4. Vinet MC
    5. Koulakoff A
    6. Carrié A
    7. Gelot A
    8. Dupuis E
    9. Motte J
    10. Berwald-Netter Y
    11. Catala M
    12. Kahn A
    13. Beldjord C
    14. Chelly J

    (1998) A novel CNS gene required for neuronal migration and involved in X-Linked subcortical laminar heterotopia and lissencephaly syndrome

    Cell 92:51–61.

    https://doi.org/10.1016/s0092-8674(00)80898-3

    • PubMed
    • Google Scholar
11. 1. Edelman AM
    2. Kim W-Y
    3. Higgins D
    4. Goldstein EG
    5. Oberdoerster M
    6. Sigurdson W

    (2005) Doublecortin Kinase-2, a novel Doublecortin-related protein kinase associated with terminal segments of axons and dendrites

    Journal of Biological Chemistry 280:8531–8543.

    https://doi.org/10.1074/jbc.M411027200

    • Google Scholar
12. 1. Edelstein A
    2. Amodaj N
    3. Hoover K
    4. Vale R
    5. Stuurman N

    (2010) Computer Control of Microscopes Using µManager

    Current Protocols in Molecular Biology 92:14–20.

    https://doi.org/10.1002/0471142727.mb1420s92

    • Google Scholar
13. 1. Ettinger A
    2. van Haren J
    3. Ribeiro SA
    4. Wittmann T

    (2016) Doublecortin is excluded from growing microtubule ends and recognizes the GDP-Microtubule lattice

    Current Biology 26:1549–1555.

    https://doi.org/10.1016/j.cub.2016.04.020

    • PubMed
    • Google Scholar
14. 1. Fan M
    2. Qian N
    3. Dai G

    (2017) Expression and prognostic significance of doublecortin-like kinase 1 in patients with hepatocellular carcinoma

    Oncology Letters 14:7529–7537.

    https://doi.org/10.3892/ol.2017.7082

    • PubMed
    • Google Scholar
15. 1. Ferguson FM
    2. Nabet B
    3. Raghavan S
    4. Liu Y
    5. Leggett AL
    6. Kuljanin M
    7. Kalekar RL
    8. Yang A
    9. He S
    10. Wang J
    11. Ng RWS
    12. Sulahian R
    13. Li L
    14. Poulin EJ
    15. Huang L
    16. Koren J
    17. Dieguez-Martinez N
    18. Espinosa S
    19. Zeng Z
    20. Corona CR
    21. Vasta JD
    22. Ohi R
    23. Sim T
    24. Kim ND
    25. Harshbarger W
    26. Lizcano JM
    27. Robers MB
    28. Muthaswamy S
    29. Lin CY
    30. Look AT
    31. Haigis KM
    32. Mancias JD
    33. Wolpin BM
    34. Aguirre AJ
    35. Hahn WC
    36. Westover KD
    37. Gray NS

    (2020) Discovery of a selective inhibitor of doublecortin like kinase 1

    Nature Chemical Biology 16:635–643.

    https://doi.org/10.1038/s41589-020-0506-0

    • Google Scholar
16. 1. Fourniol F
    2. Perderiset M
    3. Houdusse A
    4. Moores C

    (2013) Structural studies of the doublecortin family of MAPs

    Methods in Cell Biology 115:27–48.

    https://doi.org/10.1016/B978-0-12-407757-7.00003-7

    • PubMed
    • Google Scholar
17. 1. Francis F
    2. Koulakoff A
    3. Boucher D
    4. Chafey P
    5. Schaar B
    6. Vinet MC
    7. Friocourt G
    8. McDonnell N
    9. Reiner O
    10. Kahn A
    11. McConnell SK
    12. Berwald-Netter Y
    13. Denoulet P
    14. Chelly J

    (1999) Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons

    Neuron 23:247–256.

    https://doi.org/10.1016/S0896-6273(00)80777-1

    • PubMed
    • Google Scholar
18. 1. Gleeson JG
    2. Allen KM
    3. Fox JW
    4. Lamperti ED
    5. Berkovic S
    6. Scheffer I
    7. Cooper EC
    8. Dobyns WB
    9. Minnerath SR
    10. Ross ME
    11. Walsh CA

    (1998) Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein

    Cell 92:63–72.

    https://doi.org/10.1016/S0092-8674(00)80899-5

    • PubMed
    • Google Scholar
19. 1. Gleeson JG
    2. Lin PT
    3. Flanagan LA
    4. Walsh CA

    (1999) Doublecortin is a Microtubule-Associated protein and is expressed widely by migrating neurons

    Neuron 23:257–271.

    https://doi.org/10.1016/S0896-6273(00)80778-3

    • Google Scholar
20. 1. Goldberg J
    2. Nairn AC
    3. Kuriyan J

    (1996) Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I

    Cell 84:875–887.

    https://doi.org/10.1016/S0092-8674(00)81066-1

    • PubMed
    • Google Scholar
21. 1. Hornick JE
    2. Karanjeet K
    3. Collins ES
    4. Hinchcliffe EH

    (2010) Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone

    Seminars in Cell & Developmental Biology 21:290–299.

    https://doi.org/10.1016/j.semcdb.2010.01.017

    • Google Scholar
22. 1. Jean DC
    2. Baas PW
    3. Black MM

    (2012) A novel role for doublecortin and doublecortin-like kinase in regulating growth cone microtubules

    Human Molecular Genetics 21:5511–5527.

    https://doi.org/10.1093/hmg/dds395

    • Google Scholar
23. 1. Jiang D
    2. Xiao C
    3. Xian T
    4. Wang L
    5. Mao Y
    6. Zhang J
    7. Pang J

    (2018) Association of doublecortin-like kinase 1 with tumor aggressiveness and poor biochemical recurrence-free survival in prostate cancer

    Onco Targets Ther 11:1077–1086.

    https://doi.org/10.2147/OTT.S157295

    • Google Scholar
24. 1. Kadletz L
    2. Thurnher D
    3. Wiebringhaus R
    4. Erovic BM
    5. Kotowski U
    6. Schneider S
    7. Schmid R
    8. Kenner L
    9. Heiduschka G

    (2017) Role of cancer stem-cell marker doublecortin-like kinase 1 in head and neck squamous cell carcinoma

    Oral Oncology 67:109–118.

    https://doi.org/10.1016/j.oraloncology.2017.02.007

    • Google Scholar
25. 1. Kim MH
    2. Cierpicki T
    3. Derewenda U
    4. Krowarsch D
    5. Feng Y
    6. Devedjiev Y
    7. Dauter Z
    8. Walsh CA
    9. Otlewski J
    10. Bushweller JH
    11. Derewenda ZS

    (2003a) The DCX-domain tandems of doublecortin and doublecortin-like kinase

    Nature Structural & Molecular Biology 10:324–333.

    https://doi.org/10.1038/nsb918

    • Google Scholar
26. 1. Kim MH
    2. Derewenda U
    3. Devedjiev Y
    4. Dauter Z
    5. Derewenda ZS

    (2003b) Purification and crystallization of the N-terminal domain from the human doublecortin-like kinase

    Acta Crystallographica Section D Biological Crystallography 59:502–505.

    https://doi.org/10.1107/S0907444903000027

    • Google Scholar
27. 1. Kinoshita E
    2. Kinoshita-Kikuta E
    3. Koike T

    (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE

    Nature Protocols 4:1513–1521.

    https://doi.org/10.1038/nprot.2009.154

    • Google Scholar
28. 1. Koizumi H
    2. Fujioka H
    3. Togashi K
    4. Thompson J
    5. Yates JR
    6. Gleeson JG
    7. Emoto K

    (2017) DCLK1 phosphorylates the microtubule-associated protein MAP7D1 to promote axon elongation in cortical neurons

    Developmental Neurobiology 77:493–510.

    https://doi.org/10.1002/dneu.22428

    • Google Scholar
29. 1. Li L
    2. Bellows CF

    (2013) Doublecortin-like kinase 1 exhibits Cancer stem cell-like characteristics in a human Colon cancer cell line

    Chinese Journal of Cancer Research = Chung-Kuo Yen Cheng Yen Chiu 25:134–142.

    https://doi.org/10.3978/j.issn.1000-9604.2013.03.02

    • PubMed
    • Google Scholar
30. 1. Lin PT
    2. Gleeson JG
    3. Corbo JC
    4. Flanagan L
    5. Walsh CA

    (2000) DCAMKL1 encodes a protein kinase with homology to doublecortin that regulates microtubule polymerization

    The Journal of Neuroscience 20:9152–9161.

    https://doi.org/10.1523/JNEUROSCI.20-24-09152.2000

    • PubMed
    • Google Scholar
31. 1. Lipka J
    2. Kapitein LC
    3. Jaworski J
    4. Hoogenraad CC

    (2016) Microtubule-binding protein doublecortin-like kinase 1 (DCLK1) guides kinesin-3-mediated cargo transport to dendrites

    The EMBO Journal 35:302–318.

    https://doi.org/10.15252/embj.201592929

    • PubMed
    • Google Scholar
32. 1. Liu JS
    2. Schubert CR
    3. Fu X
    4. Fourniol FJ
    5. Jaiswal JK
    6. Houdusse A
    7. Stultz CM
    8. Moores CA
    9. Walsh CA

    (2012) Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins

    Molecular Cell 47:707–721.

    https://doi.org/10.1016/j.molcel.2012.06.025

    • PubMed
    • Google Scholar
33. 1. Liu Y-H
    2. Tsang JYS
    3. Ni Y-B
    4. Hlaing T
    5. Chan S-K
    6. Chan K-F
    7. Ko C-W
    8. Mujtaba SS
    9. Tse GM

    (2016) Doublecortin-like kinase 1 expression associates with breast cancer with neuroendocrine differentiation

    Oncotarget 7:1464–1476.

    https://doi.org/10.18632/oncotarget.6386

    • Google Scholar
34. Preprint

    1. Manka SW
    2. Moores CA

    (2019) Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

    bioRxiv.

    https://doi.org/10.1101/808899

    • Google Scholar
35. 1. McKenney RJ
    2. Huynh W
    3. Tanenbaum ME
    4. Bhabha G
    5. Vale RD

    (2014) Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes

    Science 345:337–341.

    https://doi.org/10.1126/science.1254198

    • Google Scholar
36. 1. Meng H
    2. Smith SD
    3. Hager K
    4. Held M
    5. Liu J
    6. Olson RK
    7. Pennington BF
    8. DeFries JC
    9. Gelernter J
    10. O'Reilly-Pol T
    11. Somlo S
    12. Skudlarski P
    13. Shaywitz SE
    14. Shaywitz BA
    15. Marchione K
    16. Wang Y
    17. Paramasivam M
    18. LoTurco JJ
    19. Page GP
    20. Gruen JR

    (2005) From The Cover: DCDC2 is associated with reading disability and modulates neuronal development in the brain

    PNAS 102:17053–17058.

    https://doi.org/10.1073/pnas.0508591102

    • Google Scholar
37. 1. Meng QB
    2. Yu JC
    3. Kang WM
    4. Ma ZQ
    5. Zhou WX
    6. Li J
    7. Zhou L
    8. Cao ZJ
    9. Tian SB

    (2013) Expression of doublecortin-like kinase 1 in human gastric Cancer and its correlation with prognosis]

    Zhongguo Yi Xue Ke Xue Yuan Xue Bao. Acta Academiae Medicinae Sinicae 35:639–644.

    https://doi.org/10.3881/j.issn.1000-503X.2013.06.010

    • PubMed
    • Google Scholar
38. 1. Mészáros B
    2. Erdos G
    3. Dosztányi Z

    (2018) IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding

    Nucleic Acids Research 46:W329–W337.

    https://doi.org/10.1093/nar/gky384

    • PubMed
    • Google Scholar
39. 1. Monroy BY
    2. Sawyer DL
    3. Ackermann BE
    4. Borden MM
    5. Tan TC
    6. Ori-McKenney KM

    (2018) Competition between microtubule-associated proteins directs motor transport

    Nature Communications 9:1487.

    https://doi.org/10.1038/s41467-018-03909-2

    • Google Scholar
40. 1. Monroy BY
    2. Tan TC
    3. Oclaman JM
    4. Han JS
    5. Simó S
    6. Niwa S
    7. Nowakowski DW
    8. McKenney RJ
    9. Ori-McKenney KM

    (2020) A combinatorial MAP code dictates polarized microtubule transport

    Developmental Cell 53:60–72.

    https://doi.org/10.1016/j.devcel.2020.01.029

    • Google Scholar
41. 1. Moores CA
    2. Perderiset M
    3. Francis F
    4. Chelly J
    5. Houdusse A
    6. Milligan RA

    (2004) Mechanism of microtubule stabilization by doublecortin

    Molecular Cell 14:833–839.

    https://doi.org/10.1016/j.molcel.2004.06.009

    • PubMed
    • Google Scholar
42. 1. Moores CA
    2. Perderiset M
    3. Kappeler C
    4. Kain S
    5. Drummond D
    6. Perkins SJ
    7. Chelly J
    8. Cross R
    9. Houdusse A
    10. Francis F

    (2006) Distinct roles of doublecortin modulating the microtubule cytoskeleton

    The EMBO Journal 25:4448–4457.

    https://doi.org/10.1038/sj.emboj.7601335

    • PubMed
    • Google Scholar
43. 1. Nagamine T
    2. Shimomura S
    3. Sueyoshi N
    4. Kameshita I

    (2011) Influence of ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

    Journal of Biochemistry 149:619–627.

    https://doi.org/10.1093/jb/mvr013

    • PubMed
    • Google Scholar
44. Software

    1. National Cancer Institute

    (2020) National Cancer Institute

    National Cancer Institute.

    https://www.cancer.gov/types
45. 1. Patel O
    2. Dai W
    3. Mentzel M
    4. Griffin MD
    5. Serindoux J
    6. Gay Y
    7. Fischer S
    8. Sterle S
    9. Kropp A
    10. Burns CJ
    11. Ernst M
    12. Buchert M
    13. Lucet IS

    (2016) Biochemical and structural insights into Doublecortin-like kinase domain 1

    Structure 24:1550–1561.

    https://doi.org/10.1016/j.str.2016.07.008

    • PubMed
    • Google Scholar
46. 1. Patel O
    2. Kropp A
    3. Dai W

    (2021) Structural basis for small molecule targeting of doublecortin like kinase 1 DCLK1

    Nature 3:133153.

    https://doi.org/10.21203/rs.3.rs-133153/v1

    • Google Scholar
47. 1. Qu D
    2. Johnson J
    3. Chandrakesan P
    4. Weygant N
    5. May R
    6. Aiello N
    7. Rhim A
    8. Zhao L
    9. Zheng W
    10. Lightfoot S
    11. Pant S
    12. Irvan J
    13. Postier R
    14. Hocker J
    15. Hanas JS
    16. Ali N
    17. Sureban SM
    18. An G
    19. Schlosser MJ
    20. Stanger B
    21. Houchen CW

    (2015) Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells

    PLOS ONE 10:e0118933.

    https://doi.org/10.1371/journal.pone.0118933

    • PubMed
    • Google Scholar
48. 1. Reiner O
    2. Coquelle FM
    3. Peter B
    4. Levy T
    5. Kaplan A
    6. Sapir T
    7. Orr I
    8. Barkai N
    9. Eichele G
    10. Bergmann S

    (2006) The evolving doublecortin (DCX) superfamily

    BMC Genomics 7:188.

    https://doi.org/10.1186/1471-2164-7-188

    • PubMed
    • Google Scholar
49. 1. Sapir T
    2. Horesh D
    3. Caspi M
    4. Atlas R
    5. Burgess HA
    6. Wolf SG
    7. Francis F
    8. Chelly J
    9. Elbaum M
    10. Pietrokovski S
    11. Reiner O

    (2000) Doublecortin mutations cluster in evolutionarily conserved functional domains

    Human Molecular Genetics 9:703–712.

    https://doi.org/10.1093/hmg/9.5.703

    • Google Scholar
50. 1. Sarkar S
    2. Popov VL
    3. O'Connell MR
    4. Stevenson HL
    5. Lee BS
    6. Obeid RA
    7. Luthra GK
    8. Singh P

    (2017) A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy

    Laboratory Investigation 97:1245–1261.

    https://doi.org/10.1038/labinvest.2017.40

    • Google Scholar
51. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
52. 1. Shang L
    2. Kwon YG
    3. Nandy S
    4. Lawrence DS
    5. Edelman AM

    (2003) Catalytic and regulatory domains of doublecortin kinase-1

    Biochemistry 42:2185–2194.

    https://doi.org/10.1021/bi026913i

    • PubMed
    • Google Scholar
53. 1. Steinmetz MO
    2. Prota AE

    (2018) Microtubule-Targeting agents: strategies to hijack the cytoskeleton

    Trends in Cell Biology 28:776–792.

    https://doi.org/10.1016/j.tcb.2018.05.001

    • PubMed
    • Google Scholar
54. 1. Sullivan LS
    2. Heckenlively JR
    3. Bowne SJ
    4. Zuo J
    5. Hide WA
    6. Gal A
    7. Denton M
    8. Inglehearn CF
    9. Blanton SH
    10. Daiger SP

    (1999) Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa

    Nature Genetics 22:255–259.

    https://doi.org/10.1038/10314

    • Google Scholar
55. 1. Taylor KR
    2. Holzer AK
    3. Bazan JF
    4. Walsh CA
    5. Gleeson JG

    (2000) Patient mutations in doublecortin define a repeated Tubulin-binding domain

    Journal of Biological Chemistry 275:34442–34450.

    https://doi.org/10.1074/jbc.M007078200

    • Google Scholar
56. 1. Westphalen CB
    2. Quante M
    3. Wang TC

    (2017) Functional implication of Dclk1 and Dclk1-expressing cells in cancer

    Small GTPases 8:164–171.

    https://doi.org/10.1080/21541248.2016.1208792

    • Google Scholar
57. 1. Weygant N
    2. Qu D
    3. Berry WL
    4. May R
    5. Chandrakesan P
    6. Owen DB
    7. Sureban SM
    8. Ali N
    9. Janknecht R
    10. Houchen CW

    (2014) Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

    Molecular Cancer 13:103.

    https://doi.org/10.1186/1476-4598-13-103

    • Google Scholar
58. 1. Wieczorek M
    2. Tcherkezian J
    3. Bernier C
    4. Prota AE
    5. Chaaban S
    6. Rolland Y
    7. Godbout C
    8. Hancock MA
    9. Arezzo JC
    10. Ocal O
    11. Rocha C
    12. Olieric N
    13. Hall A
    14. Ding H
    15. Bramoullé A
    16. Annis MG
    17. Zogopoulos G
    18. Harran PG
    19. Wilkie TM
    20. Brekken RA
    21. Siegel PM
    22. Steinmetz MO
    23. Shore GC
    24. Brouhard GJ
    25. Roulston A

    (2016) The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity

    Science Translational Medicine 8:ra159.

    https://doi.org/10.1126/scitranslmed.aag1093

    • Google Scholar
59. 1. Zhang S
    2. Zhang G
    3. Guo H

    (2017) DCAMKL1 is associated with the malignant status and poor outcome in bladder Cancer

    Tumor Biology 39:101042831770382.

    https://doi.org/10.1177/1010428317703822

    • Google Scholar

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

• 1,276

  views

• 182

  downloads

• 25

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.