Fig 1: The FRY sequence of DCX is critical for KLHL15 binding and KLHL15-mediated polyubiquitination in cells and in vitro.A, GFP-tagged KLHL15 or Drp1 was coexpressed with FLAG-tagged DCX, DCLK1, or DCLK2, either WT or FRY-mutant in COS-1 cells, and interaction was assessed by GFP-IP and immunoblotting with the indicated antibodies. B, relative DCX (or DCLK) level after IP was quantified by dividing IP'd FLAG signals to IP'd GFP signals and then to FLAG signals in the corresponding input lane (FLAGIP/GFPIP/FLAGInput) and normalizing to the WT group. Shown are means ± SD as well as individual data points from four independent experiments. ∗∗∗∗p < 0.0001 by Student t test. C, COS-1 cells coexpressing GFP-tagged KLHL15 and FLAG-tagged DCX-WT or Y259L were treated with and without the proteasome inhibitor MG132, and lysates were subjected to FLAG-IP followed by immunoblotting with the indicated antibodies. GFP-Drp1 and FLAG-RIβ were included as a negative control for GFP or FLAG protein, respectively. The unmodified DCX was detected by a short exposure (SE), whereas polyubiquitinated DCX was visualized by long exposure (LE). Shown are Western blots representative of four independent experiments. D, Myc-tagged ubiquitin and purified E1/E2 enzymes were mixed with immunoisolated Cul3–KLHL15 E3 ubiquitin ligase and bacterially expressed DCX-HaloTag-His6 protein (WT or Y259L), and in vitro ubiquitination reactions (37 °C for the indicated times) were initiated by addition of ATP. Molecular mass marker positions are shown in kilodaltons. E, the time course of in vitro DCX ubiquitination was quantified by dividing Myc signals to DCX signals and GFP signals in the same lane (Myc/DCX/GFP) and normalizing to the zero time point (set as 1). Data are shown as means ± SD from four independent experiments and were fitted to the Michaelis–Menten curve using GraphPad Prism.
Fig 2: The minimal KLHL15 docking region of DCLK2 includes both DCX domains.A, domain diagrams of DCLK2, DCLK3, and full-length or truncated DCLK chimeras. Red arrows mark the locations of the FRY sequence. B, GFP-tagged KLHL15 was coexpressed with the indicated FLAG-tagged DCLK constructs in COS-1 cells, and interactions were analyzed by GFP-IP and probing for FLAG. Shown are Western blots representative of four independent experiments. Molecular mass marker positions are shown in kilodaltons. FL, full length; Ub, polyubiquitinated species.
Fig 3: KLHL15 facilitates DCLK2 protein degradation in a Y276-dependent manner. HEK293T cells were cotransfected with DCLK2-HaloTag (WT or Y276L) and either the indicated shRNA plasmids or GFP-KLHL15. DCLK2-HaloTag was pulse labeled (30 min, 50 nM) with TMR fluorescent ligand and then chased with the non-fluorescent ligand 7-bromoheptanol for the indicated times before cell lysis. A, total lysate blots of cells expressing WT (left) or mutant (right) DCLK2-HaloTag were first imaged for TMR fluorescence and then immunoblotted for DCLK2 and GFP. Molecular mass marker positions are shown in kilodaltons. B, DCLK2 degradation was quantified by dividing TMR fluorescence by DCLK2 immunoblotting signals in the same lane (TMR/DCLK2) and normalizing to the zero time point. Data points are means ± SD from four independent transfections; half-lives (t1/2) were determined from the shown one-phase decay curve fits. C, AUC was quantified from the decay curves in B and is plotted as individual data points and means ± SD from four repeated experiments. Data were analyzed by one-way ANOVA followed by Dunnett's post hoc tests for multiple comparisons to the control shRNA group. ∗p < 0.05, ∗∗∗p < 0.001. AUC, area under the curve.
Fig 4: KLHL15 interacts with DCX, DCLK1, and DCLK2.A, search strategy for putative KLHL15 substrates. The human UniProt database was first screened for proteins containing the FRY sequence. The resulting 616 proteins were analyzed by consensus secondary structure (SS) prediction (https://npsa-prabi.ibcp.fr/) and filtered for proteins in which the FRY sequence is centered in the listed secondary structure (c, random coil; e, β-strand), yielding the 24 proteins listed in Table 1. B, on top is a multiple sequence alignment of human DCX (UniProt ID: O43602-1), DCLK1 (UniProt ID: O15075-1), DCLK2 (UniProt ID: G5E9L9) with FRY sequence in red and consensus secondary structure (cons SS) above. Because of insufficient homology, DCLK3 (UniProt ID: Q9C098) was aligned manually. Domain diagrams with red arrows indicating the FRY sequence are shown at the bottom. C, GFP-tagged KLHL15 was coexpressed with FLAG-tagged DCX, DCLK1, DCLK2, or DCLK3 in COS-1 cells, and KLHL15–substrate interaction was assessed by reciprocal immunoprecipitation (IP) via GFP or FLAG epitope tag and immunoblotting with the indicated antibodies. GFP-Drp1 and FLAG-RIβ were included as a negative control for GFP or FLAG protein, respectively. Western blots representative of four independent experiments are shown. D, bacterially expressed GST, GST-KLHL9Kelch, or GST-KLHL15Kelch protein was incubated with a whole brain lysate from E18 rat embryos, and association of endogenous proteins was evaluated by GST pulldown and immunoblotting with the indicated antibodies. Western blots representative of three independent experiments are shown. Arrows point to the full-length GST-KLHL9Kelch and GST-KLHL15Kelch proteins. Molecular mass marker positions are indicated in kilodaltons. # indicates IgG heavy chain. LE, long exposure; SE, short exposure; Ub, polyubiquitinated species.
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