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Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.
). Katanin is a heterodimer composed of two subunits, including the catalytic subunit KATNA1 (Katanin p60 ATPase-containing subunit A1), which contains the AAA+ ATPase domain, and the regulatory subunit p80 (
). The KATNA1 subunit provides the majority of observed Katanin activity because it includes domains for microtubule interaction, p80 binding, and C large subunit interaction and the AAA+ structural domain (
). SUMOylation affects protein stability, protein biochemical activity, protein–protein interactions, and transport processes into the nucleus, thereby playing an important role in regulating cellular biological functions (
). However, KATNA1 SUMOylation and its respective function in modulating neuronal processes have not been investigated.
To explore the posttranscriptional modification regulatory mechanism of KATNA1 in modulating microtubule severing and neurite outgrowth in neurons, this study tried to determine whether KATNA1 could be SUMOylated and to elucidate the detailed molecular mechanism of KATNA1 SUMOylation and the effect of this SUMOylation modification on regulating neuronal development.
). To further explore the posttranslational modification of KATNA1, we used mass spectrometry analysis of the KATNA1 interactome. Brain lysates were pulled down by GST-KATNA1, comparing with GST. The sediments were first determined by silver staining, showing large amount of pulldown sediments in the GST-KATNA1 group (Fig. 1A). Then the lanes were subjected to mass spectrometry analysis. Among the variety of interacting proteins with KATNA1, UBC9, which is an E2 SUMO-conjugating enzyme, attracted our attention (Fig. 1B). These data suggest that KATNA1 may be SUMOylated. To verify this, we purified GST, GST-SUMO1, GST-SUMO2, GST-SUMO3, and GST-KATNA1 (Fig. 1, C and D, lower panel) for GST pulldown assays using brain lysates. The Western blotting results from the GST-SUMO pulldowns showed that KATNA1 was present in the sediments for SUMO1 and SUMO2, with SUMO2 having the highest signal (Fig. 1C, upper panel). Furthermore, SUMO2 was detected in the sediments for GST-KATNA1 (Fig. 1D, upper panel). Next, we overexpressed Flag-UBC9, GFP-KATNA1, and HA-SUMO2 in 293T cells and performed immunoprecipitation (IP) with a HA antibody. As shown in Figure 1E, the results revealed that GFP-KATNA1 is detected in the IP sediments, showing the potential SUMOylated KATNA1. Furthermore, rat brain lysates were used to perform IP with the KATNA1 antibody; the results also showed that SUMO2 is present in the sediments (Fig. 1F). Cultured hippocampal neurons were immunostained with SUMO2 and KATNA1, and confocal analysis indicated that the two colocalized in the growth protrusions (Fig. 1, G and H). These data suggest that KATNA1 interacts with SUMO2 and KATNA1 could be SUMOylated.
Acetylation of microtubules promotes KATNA1-mediated severing
). To determine the effect of SUMOylation on KATNA1 function, we chose COS7 cells for microtubule severing assay, because these cells have a flat microtubule skeleton and are convenient for imaging. Simply overexpression of KATNA1 could not induce microtubule severing, and acetylation of microtubules facilitate Katanin to access microtubules for severing in fibroblasts (
). To confirm this, we initially revealed the acetylation level of microtubules in COS7; the results showed that the endogenous microtubules in COS7 were not acetylated (Fig. 2A, DMSO group). Therefore, we treated the cells with tubacin, a compound that increases the acetylation of microtubules. This treatment significantly increased the acetylation levels of the microtubules, as evidenced by immunofluorescence staining (Fig. 2, A and B). The same trend was observed by Western blotting (Fig. 2, C and D). Pretreatment of the cells with tubacin significantly increased KATNA1-mediated microtubule severing in transfected cells (circled with white line; Fig. 2, E and F). Thus, COS7 cells were all treated with tubacin for the following experiments to observe the microtubule severing by KATNA1. These data show that acetylation enhances the microtubule severing activity of KATNA1, which is consistent with a previous report (
Next, we determined the detailed molecular bias and the impact of SUMOylation on KATNA1 function. We used bioinformatics analysis to predict the potential SUMOylation sites on KATNA1. The software tools GPS-SUMO 1.0 (
) revealed the presence of three sites in the KATNA1 protein sequence, FKLD (K77), EKKE (K157), and VKAE (K330), which are consistent with the consensus SUMOylation motif (ΨKxE/D, where Ψ is a large hydrophobic amino acid and x is an arbitrary amino acid) (
). These three motifs are conserved among a variety of vertebral species (Fig. 3A). To determine the function of each site, we mutated the potential SUMOylation site lysine (K) to arginine (R) in order to eliminate SUMOylation with the smallest impact on protein sequence and property. We constructed KATNA1 single point mutants (K77R, K157R, and K330R) and a triple point mutant (Pan) and overexpressed these plasmids in COS7 cells to observe their microtubule severing ability. Immunofluorescence staining analysis showed that wildtype (WT) KATNA1 (GFP-KATNA1) significantly induced microtubule severing in transfected cells (circled with white line), with the largely decreased fluorescence signals of microtubules (Fig. 3B, WT versus Control). Pan mutation of the three potential sites abolished the severing activity of KATNA1, revealed by the rescued microtubule fluorescence signals (Fig. 3B, Pan versus WT). The K330R mutation showed the reduced activity toward microtubule severing as Pan mutation; however, the activities of K77R and K157R mutants were not changed, acting like WT KATNA1 (Fig. 3B). The relative fluorescence intensity of microtubules was quantified and shown in Figure 3C. These data indicate that K330 SUMOylation is required for the microtubule severing activity for KATNA1, mutation of this site to arginine would abolish the activity of KATNA1. To confirm this, we performed IP assays. Wildtype or K330-mutated KATNA1 (KATNA1 K330R) were co-transfected with HA-UBC9 with Flag-SUMO2 encoding plasmids, and the lysates were immunoprecipitated with a GFP antibody. The KATNA1 K330R led to a significant reduction in the level of SUMOylation (Fig. 3D; the obvious SUMOylated bands indicated by arrow). Wildtype KATNA1 (GFP-KATNA1 WT) interacted with SUMO2 (Fig. 3E), but the significantly reduced signals from the sediments of K330R revealed that the KATNA1 K330R mutant disrupted the interaction between KATNA1 and SUMO2 (Fig. 3F). These data indicate that K330 is an essential residue for KATNA1 SUMOylation and SUMOylation of this residue has a direct effect on KATNA1-mediated microtubule severing activity.
SUMOylation promotes the microtubule severing activity of KATNA1
To further determine the role of K330 SUMOylation in KATNA1-mediated microtubule severing activity, we overexpressed mCherry-SUMO2 and Flag-UBC9 plasmids with different GFP-KATNA1 constructs in COS7 cells. Using immunofluorescence assays (Fig. 4A, circled cells indicating the transfected cells), we observed that overexpression of WT KATNA1 significantly induced microtubule severing relative to the control group or SUMO2 alone group, and co-transfection of KATNA1 with SUMO2 further increased the observed severing efficiency. However, KATNA1 K330R could not sever microtubules even when cotransfected with SUMO2 (Fig. 4B). These data further confirm that K330-mediated SUMOylation of KATNA1 promotes its microtubule severing activity.
Suppression of SUMOylation of KATNA1 abolishes the promoting effect in hippocampal neurite outgrowth
Next, we determined the role of KATNA1 SUMOylation in hippocampal neurite outgrowth. Because KATNA1 favors an acetylated microtubule cytoskeleton, we first examined the acetylation level of the hippocampal microtubules. As shown in Figure 5A, strong immunofluorescence signals were observed for acetylated microtubules in 2 to 4 DIV (days in vitro) neurons. To further understand the effect of KATNA1 SUMOylation on neurite outgrowth in hippocampal neurons, DIV2 primary hippocampal neuronal cells were sequentially transfected with GFP, GFP-KATNA1, and the mutants (Fig. 5B). The results showed that total length, axonal length, and dendritic length per neuron were significantly increased in KATNA1 WT and K77R and K157R overexpressing groups, but not in K330R and Pan mutant groups (Fig. 6, A–C). The number of tips (branches, Fig. 6, D–F) and neuronal complexity revealed by Sholl analysis (Fig. 6,G–I) showed the same trend as neurite length. These data demonstrate that KATNA1 promotes hippocampal neurite outgrowth, and the effect can be abolished if SUMOylation is absent or suppressed by mutation.
SUMOylation of KATNA1 enhances hippocampal neurite outgrowth
To further confirm the effect of KATNA1 SUMOylation on hippocampal neurite outgrowth, we overexpressed HA-UBC9 with or without WT or K330R GFP-KATNA1 or Flag-SUMO2. As shown in Figure 7, relative to the control or SUMO2 groups, overexpression of WT KATNA1 markedly promoted neurite outgrowth, with a concurrent increase in the neurite length (Fig. 8, A–C), the number of tips (Fig. 8, D–F), and neuronal complexity (Fig. 8,G–I). Moreover, co-overexpression of WT KATNA1 and SUMO2 further increased neurite outgrowth, whereas co-overexpression of K330R and SUMO2 exhibited no effects (Fig. 8). These data suggest that SUMOylation enhances the promoting effect of KATNA1 in regulating neurite outgrowth.
In the current study, we show that KATNA1 is SUMOylated at K330. Disruption of this SUMOylation by mutation abolishes KATNA1 activity, whereas increased SUMOylation enhances the microtubule severing activity of KATNA1 and neurite outgrowth. The schematic diagram of this regulatory mechanism is shown in Figure 9. Our data supplement the landscape of KATNA1 regulation by posttranslational modification, which might play an important role in microtubule-related neuronal diseases.
SUMOylation is involved in the progression of numerous diseases, including cancer and Huntington’s, Alzheimer’s, and Parkinson’s diseases (
). For example, the K293 residue in low-density lipoprotein receptor (IDOL) is modified by both SUMOylation and ubiquitination, wherein SUMOylation can counteract ubiquitination to increase the levels of IDOL (
). The deubiquitinating enzyme ubiquitin carboxy-terminal hydrolase 37 (USP37) is modified by SUMOylation and ubiquitination at different sites, and SUMOylation at K452 promotes subsequent ubiquitination of K148 and K389 (
). Katanin can be ubiquitinated. Antagonistic regulation of KATNA1 activity by E3 ubiquitin-protein ligase CHIP (CHIP) and the deubiquitinating enzyme ubiquitin carboxyl-terminal hydrolase 47 (USP47) influences axonal growth in hippocampal neurons (
). In the present study, SUMOylation of KATNA1 promoted the development of neuronal protrusions. Whether or not K330 is ubiquitinated and the relationship between KATNA1 SUMOylation and ubiquitination need to be investigated further.
The stability and activity of Katanin are affected by phosphorylation (
). Hence, there is cross talk between phosphorylation and SUMOylation. How phosphorylation events affect KATNA1 SUMOylation (or the reverse) should be examined in future studies.
Protein interactions regulate KATNA1 activity; for example, abnormal spindle-like microcephaly-associated protein recruits KATNA1 and P80 to the negative end of microtubules, thereby limiting their growth during mitosis (
). Cyclin-dependent kinase 5 phosphorylation of nuclear distribution protein nude-like 1 (NDEL1) promotes its interaction with KATNA1, which regulates the localization and activity of KATNA1 during neuronal migration (
). Calmodulin-regulated spectrin-associated proteins (CAMSAPs) are deposited at the negative end of microtubules to avoid spontaneous microtubule depolymerization, and Katanin binds to CAMSAPs to synergistically limit microtubule extension and maintain microtubule stability (
). Therefore, interaction with proteins regulates the activity of Katanin through different pathways. Our findings that indicate that KATNA1 is SUMOylated and that SUMOylation enhances the microtubule cleaving activity of KATNA1 are supported in the literature. KATNA1 and Spastin are both in the AAA protease family and may regulate microtubule dynamics in a similar manner (
). Previously, we demonstrated that SUMOylation promotes maturation of neuronal dendritic spines and transport of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors. In addition, mutation of the Spastin SUMOylation site K427 abolishes its ability to cleave microtubules (
). The results from these studies indicate that mutation of SUMOylation sites not only reduces the level of SUMOylation but also inhibits protein function.
Overall, we identified a novel mechanism regarding the SUMOylation of KATNA1 at K330 that promotes microtubule severing and neurite outgrowth, which provides new insight into the regulation of KATNA1 activity that may lead to novel therapeutic treatments for microcephaly and other neuronal developmental disorders.
). The KATNA1 (AY621629.1), SUMO1 (NM_003352.8), SUMO2 (NM_001005849.2), SUMO3 (NM_006936.3), and UBC9 (BC000427.2) genes were cloned from rat cDNA and validated via sequencing. SUMO1 and SUMO3 cDNA were inserted into a pGEX-5x-3 vector (MiaoLingBio, cat. no. #P0004), and SUMO2 was subcloned into pGEX-5x-3, pLVX-mCherry-N1 (Takarabio, cat. no. #632562), pCMV-HA (Takarabio, cat. no. #635690), and pCMV-FLAG-C (MiaoLingBio, cat. no. #P1003). KATNA1 was inserted into pEGFP-C1 (MiaoLingBio, cat. no. #P0134). UBC9 was inserted into pCMV-FLAG-C and pCMV-HA. The corresponding mutant plasmids (K77R, K157R, K330R, and Pan) for GFP-KATNA1 were constructed using point mutation kits (Invitrogen, cat. no. #A14606). All constructs were verified after sequencing.
Protein purification and GST pulldown assays
Protein expression and GST pulldown assays were described (
). Briefly, GST-SUMO1, GST-SUMO2, and GST-SUMO3 were transformed into BL21 pLysS chemically competent cells (TransGen, cat. no. #CD701-02). Bacteria were cultured in 250 ml of Luria–Bertani medium (Sigma, cat. no. #L3152) containing ampicillin (Sigma, cat. no. #A5354) and were induced with 0.1 mM IPTG (Sigma, cat. no. #I6758) for 8 h at 30 °C. The bacteria were collected and 12.5 ml of lysis buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.0, and 5% glycerol), 1% Triton X-100, and a cocktail of protease inhibitors (Merck, cat. no. #HY-K100) consisting of 1 μM phenylmethylsulfonyl fluoride (Merck, cat. no. # PMSF-RO) were added. The bacteria were lysed on ice using sonication at 20% intensity for 1 h (every 15 min, the lysate was sonicated for 1 min, shaken for 30 s, and incubated on ice). After centrifugation of the lysate, 200 μl of glutathione agarose beads (Pierce Biotechnology, cat. no. #17-0756-01) were added to the supernatant. This mixture was inverted and mixed overnight at 4 °C, and the beads were retained after centrifugation. GST-fused protein (30–50 μg) was mixed with rat brain lysate, inverted overnight at 4 °C, and finally verified using Western blotting.
Cell culture and transfection
Primary hippocampal neurons were cultured as described (
). P0 (1-day-post-birth) Sprague Dawley rats were sacrificed by decapitation and then the hippocampus was taken from the brains. A total of 80 rat pups were used in this study, 3 pups were used for each hippocampal culture. All rats were purchased from Southern Medical University (Production license: SCXK (Guangdong) 2016-0041, Use license: SYXK (Guangdong) 2016-016). All animal procedures were approved using the Guide for the Care and Use of Laboratory Animals from the NIH and were approved by the Jinan University Institutional Animal Care and Use Committee (Approval no. 20190310-05). The hippocampus was placed in 0.125% trypsin and was incubated in a 37 °C water bath for 20 min. Trypsin digestion was terminated with Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, cat. no. #11995065) with 10% fetal bovine serum (Gibco, cat. no. #10270-106). The cells were plated on glass coverslips coated with poly-D-lysine (Sigma, cat. no. #P6407) at a density of 1 × 104 cells/cm2. After incubation for 30 min, 500 μl of DMEM/F12 with 10% fetal bovine serum was added to each well. The medium was replaced with neurobasal media supplemented with 2% B27 (Gibco, cat. no. #17504044) after 3 to 4 h. Cells were cultured at 37 °C with 5% CO2. The plasmids were transfected at 2 DIV (days in vitro) using a calcium phosphate cell transfection kit (Beyotime, cat. no. #C0508) and were incubated for 48 h. The cells were fixed using 4% paraformaldehyde (Meilunbio, cat. no. #MA0192) for the immunofluorescence experiments.
COS7 and 293T cells were purchased from the National Collection of Authenticated Cell Cultures (STR certified), within five passages. The cells were cultured in DMEM medium (Gibco, cat. no. #C11995500BT) with 10% fetal bovine serum at 37 °C with 5% CO2; the cells were transfected using lipofectamine 2000 (Invitrogen, cat. no. #11668019).
). Briefly, COS7 cells or hippocampal neurons were fixed using 4% paraformaldehyde for 40 min at 4 °C. The cells were then washed twice with TBS (Meilunbio, cat. no. #MA0141) with 0.1% Triton (TBST, Meilunbio, cat. no. #MB2486) at RT. The cells were blocked for 1 h at RT with 3% bovine serum albumin (Sigma, cat. no. #V900933-100G) in TBST. The cells were incubated with primary antibodies (GFP [Abcam, cat. no. #ab290, 1:1000], beta-Tubulin [Abcam, cat. no. #ab6046, 1:1000, for general tubulin staining], acetylated tubulin [Abcam, cat. no. #ab24610, 1:1000], KATNA1 [Proteintech, cat. no. #17560-1-AP, 1:200], MAP-2 [Abcam, cat. no. #ab5392, 1:500], Flag [Sigma, cat. no. #F9291, 1:3000], or SUMO2 [Novus, cat. no. #NBP2-44477, 1:1000]) at 4 °C overnight. Cells were washed three times and incubated with the corresponding secondary antibody (Thermo Fisher Scientific; Alexa Fluor 488, cat. no. #A-10680, RRID: AB 2534062; Alexa Fluor 555, cat. no. #A-21428, RRID: AB_2535849; Abcam, Alexa Fluor 647, cat. no. #ab150159) at RT for 1 h in the darkroom. The cells were fixed with NucBlue Fixed Cell ReadyProbes Reagent (DAPI, Electron Microscopy Science, cat. no. #17985-50) and imaged via confocal microscopy with an LSM 880 (Carl Zeiss).
). Briefly, 293T cells were lysed using lysis buffer (Beyotime, cat. no. #P0013). Equal amounts of total protein were loaded and run on a 10% SDS-PAGE gel and was transferred to a polyvinylidene fluoride membrane (Millipore, cat. no. #IPVH00010) for 1 h. The polyvinylidene fluoride membrane was blocked for 1 h at RT in TBS with 0.1% Tween-20 (TBST, Meilunbio, cat. no. #MB2483) and 5% skim milk (BD Difco, cat. no. #232100-500G). The membranes were incubated with primary antibodies (GFP [Abcam, cat. no. #ab290, 1:1000], HA [Sigma, cat. no. #H9658, 1:1000], KATNA1 [Proteintech, cat. no. #17560-1-AP, 1:500], beta-Tubulin [Abcam, cat. no. #ab6046, 1:5000], Flag [Sigma, cat. no. #F9291, 1:1000], GAPDH [Sigma, cat. no. #G9545, 1:5000], or acetylated tubulin [Abcam, cat. no. #ab179484, 1:5000]) at 4 °C overnight; the membranes were washed three times with TBST and incubated with the corresponding secondary antibody (1:5000, Abclonal Biotechnology, HRP Goat Anti-mouse IgG (H + L), cat. no. #AS003; HRP Goat Anti-rabbit IgG (H + L), cat. no. #AS014; HRP Goat Anti-rat IgG (H + L), cat. no. #AS028) at RT for 1 h. Membranes were exposed to BeyoECL Plus (Beyotime, cat. no. #P0018S) for visualization.
). Briefly, GFP-overexpressed hippocampal neurons were immunostained with antibodies against GFP for total neurite and MAP2 for dendrites, then neurons were imaged in a blinded manner with Axio Observer Z1 (Carl Zeiss). The parameters of total neurite length per neuron, axonal/dendritic length per neuron, total tips per neuron, axonal/dendritic tips per neuron from at least 20 neurons per group were calculated by Image Pro Plus 6 software (Media Cybernetics).
). Briefly, the images were randomly collected using an LSM 880 confocal microscope (Carl Zeiss), and each group contained at least 20 neurons. ImageJ (NIH) was used to change the image to 8-bit grayscale, and the modified images were saved. The saved image was opened in NeuronJ (
), the neuron trajectory was drawn. Taking the neuron cell body as the center, a circle was drawn with a radius that contained all the protrusions, and this image was used for subsequent calculations. Data were exported to GraphPad Prism 5 for measurement. The groups were blinded for measurement and then unblinded for group labels.
Mass spectrometry was used to analyze sediments from GST-KATNA1 pulldown assays. The gel was digested with trypsin. GST and GST-KATNA1 samples were used for nano-liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. After chromatographic separation, the samples were analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific) for 60 min. The detection method was positive ion mode, the precursor ion scan range was 300 to 1800 m/z, the resolution of the primary mass spectrum was 70,000 at 200 m/z, the automatic gain control target was 1e6, the Maximum IT was 50 ms, and the dynamic exclusion time (Dynamic exclusion) was 60 s. MS/MS spectra were searched against the UniProtKB database using the MASCOT engine (Matrix Science; version 2.4).
All data are presented as the means ± SD from at least three independent experiments. GraphPad Prism 7 was used to generate graphs and statistics; ImageJ (NIH) was used to measure the microtubule fluorescence intensity in COS7 cells. All data in this study used D'Agostino and Pearson normality test for normality test. For data that conform to the normal distribution, analyses were performed using a t test or one-way analysis of variance (ANOVA). For data that do not conform to a normal distribution, a nonparametric test (Wilcoxon rank sum test) was applied. All tests are two-tailed tests. p < 0.05 indicates statistical significance, (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
This work was supported by the National Natural Science Foundation of China (nos. 32071033 , 81771331 , and 32170977 ).
G. Z., M. T. conceptualization; Y. L. methodology; J. Z. software; Z. C., H. Y., J. Y., Y. Z. validation; S. L., Y. L., Z. C. data curation; S. L. writing – original draft; H. Y., J. Y., Y. Z., H. L., G. Z., M. T. writing – review & editing; J. Z. visualization; M. T. supervision; M. T. project administration.
Cutting, amplifying, and aligning microtubules with severing enzymes.