Biophysical Journal | 2021
Autoregulatory Control of Microtubule Binding in Doublecortin-Like Kinase 1
Abstract
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 found 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 has 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. Introduction 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 Agulto et al. eLife 2021;10:e60126. DOI: https://doi.org/10.7554/eLife.60126 1 of 22 RESEARCH ARTICLE 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. 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. Results 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 interor 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 (DC: 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-DC proteins migrated more slowly into the Phos-tag Agulto et al. eLife 2021;10:e60126. DOI: https://doi.org/10.7554/eLife.60126 2 of 22 Research article Biochemistry and Chemical Biology Cancer Biology