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. 2024 Mar 25;38(7):e25030. doi: 10.1002/jcla.25030

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns

Weiwen Bu 1, Jie Di 1, Junkui Zhao 1, Ruming Liu 1, Yue Wu 2, Jie Ran 2,✉, Te Li 1,✉
PMCID: PMC11033342  PMID: 38525916

ABSTRACT

Background

The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo‐specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions.

Methods

Using bioinformatics, we analyzed the C‐terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post‐translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site‐directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex.

Results

We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1).

Conclusions

The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein‐related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

Keywords: arginine methylation, dynein, light intermediate chain, microtubule, PRMT1

Using bioinformatics, we analyzed the C‐terminal domains of LIC1 and LIC2, revealing diverse post‐translational modifications (PTMs). The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently.

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1. Introduction

Intracellular transport is a marvel of biological precision, involving the carefully orchestrated movement of membrane vesicles and organelles along the cellular scaffold [1]. This process relies predominantly on microtubule‐based motors, such as kinesins and dynein, which move cargo with spatiotemporal accuracy and efficiency [2, 3, 4, 5]. Kinesins typically move toward the plus‐end of microtubules, while cytoplasmic dynein predominates in transporting materials toward the minus‐end [6]. Despite the elegance of this system, the mechanisms by which membrane cargoes select appropriate motors and coordinate the directionality of movements remain a crucial knowledge gap in our understanding of intracellular traffic.

Emerging research has shed light on these enigmas with the discovery of selective microtubule post‐translational modifications and microtubule‐associated proteins tailored for the transit of specific motor–cargo complexes [7]. Furthermore, cargo adaptor and scaffold proteins have been identified as key regulators of motor–cargo interactions, playing a central role in the modulation of motor binding and activity [8].

Dynein, a homodimeric multi‐protein complex, is composed of two heavy chains and various accessory subunits, including the intermediate, light, and light intermediate chains (LICs) [9]. Essential for a host of cellular functions ranging from protein and RNA transport to nuclear positioning and cell division, dynein's interaction with different cargoes has been clarified by the identification of specific dynein adaptors [10]. These adaptors are pivotal for recruiting the dynactin regulatory complex, thereby enhancing dynein's processivity, velocity, force production, and cargo diversity [11, 12]. Of note, the LICs have emerged as critical determinants that engage with the adaptors [13, 14, 15]. Vertebrate evolution has resulted in three LIC variants: LIC1 (DYNC1LI1), LIC2 (DYNC1LI2), and LIC3 (DYNC2LI1) [16]. While LIC1 and LIC2 are integral to cytoplasmic dynein 1 and share overlapping cellular responsibilities, LIC3 is exclusively associated with cytoplasmic dynein 2, which is implicated in ciliary transport [17, 18]. Similar to numerous microtubule‐binding proteins, LICs also have the capability to influence the spindle orientation [19, 20, 21, 22]. LIC2‐dynein disrupts stages of the spindle assembly checkpoint (SAC) that monitor microtubule attachment and inter‐kinetochore tension, whereas LIC1‐dynein preferentially targets the microtubule attachment detection stage of the SAC [23, 24].

The intricate process of post‐translational modifications such as arginine methylation significantly expands proteomic diversity and regulates many essential, dynamic cellular processes [25, 26, 27, 28, 29, 30]. Arginine methylation, a prevalent post‐translational modification in eukaryotes, is implicated in DNA repair, transcription, receptor trafficking, and the stabilization of proteins [31]. There are three recognized states of methylated arginine: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). The synthesis of these derivatives is orchestrated by protein arginine methyltransferases (PRMTs), a family of nine members [32]. PRMT1 is the most dominant, responsible for 85% of PRMT activity; deficiencies in PRMT1 have been linked to a host of diseases, including embryonic abnormalities, cancer, and neurodegenerative disorders [33, 34, 35]. For example, PRMT1 depletion leads to reduced expression of certain progesterone‐responsive genes that drive breast cancer cell proliferation and movement [36]. Additionally, PRMT1 plays a role in DNA replication by methylating MRE11, which is critical for telomere replication and the S‐phase checkpoint in response to DNA damage [37, 38].

In this study, we identified a novel target of PRMT1, LIC2, which undergoes monomethylation. The specific site of arginine methylation on LIC2 is pinpointed at residue 397. This discovery not only enriches our knowledge of the regulatory mechanisms influencing LIC2's function but also opens avenues for therapeutic interventions by manipulating its methylation status to correct or enhance intracellular transport abnormalities.

2. Materials and Methods

2.1. Cell Culture

The human bone osteosarcoma epithelial cells (U2OS) and human embryonic kidney HEK293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS). Culture conditions were optimized for growth by keeping cells at a constant temperature of 37°C in a humidified incubator with an atmospheric composition of 5% CO2.

2.2. Transfection of Plasmids and siRNAs

For plasmid transfection, HEK293 cells were seeded in plates in DMEM with 10% FBS and without antibiotics. After cell adherence, the transfection mixture containing plasmids and polyethylenimine (PolyScience) was added into the culture medium. The medium was changed to fresh DMEM with 10% FBS 12 h after transfection. For siRNA transfection, the transfection mixture containing siRNAs and Lipofectamine RNAiMAX (Invitrogen) was added into the culture medium without FBS or antibiotics. The medium was changed to fresh DMEM with 10% FBS 8 h after transfection. The LIC1 and LIC2 cDNA were cloned into the pCMV‐HA‐C vector. The arginine to alanine mutations in LIC2 were generated by PCR and site‐directed mutagenesis. The PRMT1 cDNA was cloned into the pcDNA3.1‐FLAG vector. The sequences of siRNAs used in this study were as follows: siControl, 5′‐CGUACGCGGAAUACUUCGA‐3′; siPRMT1#1, 5′‐GCAACTCCATGTTTCATAA‐3′; siPRMT1#2, 5′‐AGACGGTGTTCTACATGGA‐3′.

2.3. Immunoprecipitation and Immunoblotting

Cell lysates were prepared by solubilizing cells in lysis buffer (Bryotime) with a protease inhibitor cocktail (Thermo Fisher Scientific). Following a 30 min incubation period on ice, the lysates were subjected to centrifugation at 12,000 rpm at 4°C for 20 min. For the immunoprecipitation procedure, the clarified supernatants were incubated with antibody‐coated agarose beads for overnight at 4°C. After the incubation, the beads were washed five times, and the immunocomplexes were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and subsequently analyzed through western blotting.

For the western blot analysis, proteins separated by SDS‐PAGE were transferred onto nitrocellulose membranes (PALL BioTrace, 66485). The membranes were then blocked in a Tris‐buffered saline solution containing 0.1% Tween 20 and 5% nonfat milk (Becton, Dickinson and Company, Catalog No. 232100) for 2 h at room temperature. This was followed by incubation with specific primary antibodies and then detected with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Solarbio, Catalog No. SE134; dilution ratio of 1:5000). Enhanced chemiluminescence was carried out using the HRP substrate luminol solution (Millipore, Catalog No. WBKLS0500), with protein bands visualized under appropriate detection conditions. Primary antibodies for immunoblotting were as follows: rabbit polyclonal antibody against HA (Abcam, ab9110; dilution: 1:1000), rabbit monoclonal antibody against FLAG (Abcam, ab205606; dilution: 1:1000), mouse monoclonal antibody against α‐tubulin (Abcam, ab7291; dilution: 1:1000), rabbit monoclonal antibody against PRMT1 (Abcam, ab190892; dilution: 1:1000), rabbit monoclonal antibody against MMA (CST, 8015S; dilution: 1:1000).

2.4. Immunofluorescence

For immunofluorescence assays, cells cultured on glass coverslips were fixed using a 4% paraformaldehyde solution in phosphate‐buffered saline (PBS) for 15 min to preserve cellular architecture and protein epitopes. Following fixation, cells underwent permeabilization with a 0.5% Triton X‐100 solution in PBS for 10 min to permit antibody access to intracellular structures. Subsequently, cells were blocked with a 4% bovine serum albumin (BSA) solution in Tris‐buffered saline with Tween 20 (TBST) to minimize nonspecific antibody binding. Primary antibodies were then applied to target specific antigens, after which cells were incubated with fluorescently labeled secondary antibodies conjugated to either FITC (fluorescein isothiocyanate) or rhodamine, to enable visualization of the primary antibody binding. Finally, cell nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) to facilitate the identification of cellular localization through fluorescence microscopy. Antibodies and chemicals used in this study included rabbit polyclonal antibody against HA (Abcam, ab9110; dilution: 1:1000), mouse monoclonal antibody against α‐tubulin (Abcam, ab7291; dilution: 1:1000).

3. Results

3.1. Comparative Analysis of Post‐Translational Modification (PTM) Sites in LIC C‐Terminal Domains

The N‐terminal domain (NTD) of cytoplasmic dynein light intermediate chain (LIC) adopts a G protein fold, which is instrumental in making close contacts with the tail of dynein heavy chains [39]. The functional diversity of LICs is conferred by their ability to bind a myriad of structurally diverse adaptors through their C‐terminal domains (CTDs, Figure 1A). Through sequence alignment investigations between the CTDs of LIC1 and LIC2, it was noted that these domains display approximately 59% sequence similarity (Figure 1B).

FIGURE 1.

FIGURE 1

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Different PTMs in LIC CTDs. (A) A schematic illustration of the prototypical mammalian LIC isoforms (LIC1 and LIC2) anchored to the heavy chain through their structured domains (G‐domain for the LIC NTDs). (B) Sequence alignment between CTDs of LIC1 (residues 381–523) and LIC2 (375–492). Colors indicate variability: black for the same, blue for similar, and red for different. (C) Demonstration of post‐translational modifications of LIC CTDs via websites https://www.phosphosite.org/homeAction.

Proteomic analysis has highlighted that both LIC1 and LIC2 are substrates for various PTMs at multiple sites including phosphorylation, ubiquitylation, methylation, and acetylation. Noteworthy differences have, however, been identified in the distribution of these PTM sites between the two proteins' CTDs. Specifically, the CTD of LIC1 harbors sites of acetylation, whereas such sites have not been identified in the LIC2 CTD. In contrast, the LIC2 CTD is enriched with a greater number of monomethylation sites in comparison with that of LIC1 (Figure 1C). These findings suggest that distinct regulatory mechanisms may target each LIC CTD. Moreover, the additional monomethylation sites present in LIC2 hint at potential roles for arginine methylation in the modulation of LIC2 functions, supporting the hypothesized existence of such PTMs as predicted through computational bioinformatics analyses.

3.2. The Arginine Methylation Status of LIC Proteins

Building on our structural insights, we sought to determine the methylation status of arginine residues within the LIC1 and LIC2 proteins. We transfected HEK293 cells with HA‐LIC1, HA‐LIC2, or HA vector, and the cellular extracts were subsequently subjected to immunoprecipitation using antibody against HA. The isolated immunoprecipitates were analyzed via immunoblotting employing a mono‐methyl arginine (MMA)‐specific antibody to assess the presence of arginine methylation on LIC1 and LIC2. Intriguingly, this analysis revealed that LIC2 exhibited arginine methylation, whereas LIC1 did not (Figure 2A,B).

FIGURE 2.

FIGURE 2

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LIC2 is arginine methylated. (A) HEK293 cells were transfected with HA vector or HA‐LIC1. Cell lysates were immunoprecipitated with the HA antibody, and methylation was examined with the MMA antibody. (B) HEK293 cells were transfected with HA vector or HA‐LIC2. Cell lysates were immunoprecipitated with the HA antibody, and methylation was examined with the mono‐methyl arginine (MMA) antibody. (C) HEK293 cells transfected with HA‐LIC2, and tretment with or without 40 μM AdOx (a methylation inhibitor) for 16 h. Cell lysates were immunoprecipitated with the HA antibody, and methylation was checked with the MMA antibody.

To further investigate the methylation dynamics, we utilized oxidized adenosine (AdOx), known to inhibit methylation processes [40]. Treatment with 40 μM AdOx, an established effective concentration in cell culture, led to a reduction in the detected arginine methylation levels of LIC2 (Figure 2C). This experiment substantiates arginine methylation occurring specifically on LIC2, but not on LIC1, thus highlighting a differential post‐translational regulatory mechanism that could influence the functional interplay between these closely related proteins.

3.3. LIC2 Interacts With PRMT1

PRMT1 stands as the predominant protein arginine methyltransferase in mammals and is responsible for orchestrating the monomethylation as well as asymmetric dimethylation of arginine residues within proteins [41]. PRMT1 specifically targets arginine residues nestled within glycine/arginine‐rich motifs, notably those conforming to RGG or RXR sequences [42, 43]. It is noteworthy that, among the LIC proteins, only LIC2 contains the requisite RGG motifs, hinting at a selective interaction potential.

To empirically validate the selective affinity between PRMT1 and the LIC proteins, HEK293 cells were transfected with HA‐LIC1 or HA‐LIC2, together with Flag‐PRMT1. The immunoprecipitation followed by subsequent immunoblotting analysis provided compelling evidence that PRMT1 robustly associates with LIC2 but not LIC1 (Figure 3A,B, Figure S1A,B). These results not only confirm the existence of a protein–protein interaction between LIC2 and PRMT1 but also suggest a potential specificity whereby PRMT1 preferentially methylates LIC2 over LIC1, presumably due to the presence of RGG motifs.

FIGURE 3.

FIGURE 3

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LIC2 interacts with PRMT1. (A) HEK293 cells were transfected with Flag‐PRMT1, together with HA vector or HA‐LIC2 for 24 h. Immunoprecipitation assays were performed with the HA antibody. The immunoprecipitates were subjected to SDS‐PAGE and immunoblot analysis with the Flag and HA antibodies. (B) HEK293 cells were transfected with HA‐LIC2, together with Flag vector or FLAG‐PRMT1 for 24 h. Immunoprecipitation assays were performed with the Flag antibody. The immunoprecipitates were subjected to SDS‐PAGE and immunoblot analysis with the HA and Flag antibodies.

3.4. PRMT1 Methylates LIC2

We then investigated whether LIC2 is arginine‐methylated by PRMT1. The HEK293 cells were transfected with Flag vector or Flag‐PRMT1, together with HA‐LIC2, and results demonstrated that the overexpression of PRMT1 promotes the methylation of LIC2 (Figure 4A). Conversely, the targeted downregulation of PRMT1 via siRNA‐mediated gene silencing, employing two distinct siRNA sequences, caused a remarkable diminished methylation level of LIC2 (Figure 4B). Consistently, treatment with Furamidine dihydrochloride, a specific antagonist for inhibiting PRMT1 activity, leads to a marked diminution in LIC2 methylation level (Figure 4C). These results further substantiate the role of PRMT1 in catalyzing the methylation of arginine residues within LIC2.

FIGURE 4.

FIGURE 4

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PRMT1 promotes the methylation of LIC2. (A) The methylation of LIC2 was analyzed by immunoprecipitation with the anti‐HA antibody followed by immunoblotting in cells transfected with Flag vector or Flag‐PRMT1, in the presence of HA‐LIC2. (B, C) Immunoprecipitation and immunoblotting analysis of methylation of LIC2 in cells transfected with control or PRMT1 siRNAs in the presence of HA‐lic2 (B), and in cells transfected with HA‐LIC2 with or without Furamidine (20 or 40 μM, C).

Together, these observations bolster the hypothesis that PRMT1 acts as a principal orchestrator in the arginine methylation of LIC2, thereby contributing to our understanding of the sophisticated regulatory mechanisms that govern cellular functionality through selective post‐translational modifications.

3.5. LIC2 Methylation Occurs at Arginine 397

Numerous proteomics studies have demonstrated methylation occurring at the arginine 397 site of LIC2. Sequence comparison revealed that the arginine 397 site of LIC2 is highly conserved in vertebrates. In mammals, the arginine 397 site of LIC2 aligns with the prevalent catalytic motif “RGG” of PRMT1 (Figure 5A). To determine whether the arginine 397 site is the methylation site of LIC2, we employed precision point mutation strategies to engineer a LIC2 variant, where the native arginine at the 397 was substituted with lysine (R397K). The HEK293 cells were then transfected with HA‐LIC2 wild‐type or R397K mutant, and the immunoprecipitation followed by subsequent immunoblotting analysis was performed. We observed that the R397K mutant showed a compelling methylation‐null phenotype, indicating a disrupted methylation site absent the native arginine (Figure 5B).

FIGURE 5.

FIGURE 5

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PRMT1‐mediated methylation occurs at arginine 397. (A) Protein sequence comparison of LIC2 R397 and adjacent amino acids in various species. The black‐highlighted amino acids are conserved between all species compared, and the red asterisk mark indicates the arginine 397 site of LIC2. (B) HEK293 cells were transfected with HA vector, HA‐LIC2 wild‐type, or HA‐LIC2‐R397K. Analysis of the methylation by immunoprecipitation with the HA antibody followed by immunoblotting with the MMA antibody. (C) U2OS cells were transfected with HA‐LIC2 wild‐type or the R397K mutant, g stained with antibodies against HA (green) and α‐tubulin (red). Scale bar, 10 μm.

We then investigated the effect of methylation on cellular localization of LIC2. The U2OS cells were transfected with LIC2 wild‐type or the R397K mutant. Immunostaining was performed using antibodies targeted against α‐tubulin and HA to ascertain the co‐localization of LIC2 with the microtubule network. The examination revealed that both the wild‐type and R397K mutant LIC2 retained robust co‐localization with the microtubule structures, as the patterns were indistinguishable (Figure 5C). This finding suggests that methylation modifications at the arginine 397 site do not exert a significant influence on the microtubule targeting affinity of LIC2, indicating that the methylation status of LIC2 is not a requisite determinant for its spatial organization within the cytoskeletal framework.

4. Discussion

Arginine methylation is a widespread post‐translational modification that influences a variety of cellular pathways, including signal transduction, gene expression, and DNA repair [44]. PRMT1, as a major arginine methyltransferase, is crucial for these processes and has been linked to several human diseases [45, 46, 47]. Our study unearths a novel revelation—identifying LIC2 as a previously unrecognized substrate of PRMT1. This discovery enriches the discourse on PRMT1's repertoire of activity, offering new depth to its functional narrative.

Despite having partially distinct functional roles, LIC2 has received relatively limited characterization compared with its vertebrate homolog LIC1. These closely related homologs are believed to operate in mutually exclusive complexes, carrying out distinct yet complementary functions [48]. During the interphase in mammalian cells, LIC1‐dynein is involved in transporting pericentrin to the centrosomes, organizing Golgi vesicles at the nuclear periphery, and governing the distribution of lysosomes and late endosomes to a greater extent than LIC2 [49, 50, 51]. In the development of the rat brain, LIC1‐dynein aids in the apical migration of cell nuclei in neural precursors, drives the transition to bipolar morphology in emerging neurons, and directs neuron migration via glial guidance, unlike LIC2‐dynein, which is singularly tied to positioning cell bodies in their final brain locations [52]. In contrast, during mitosis, LIC1 plays a more significant role in maintaining spindle pole integrity, while LIC2 has been shown to regulate spindle orientation through interactions with 14‐3‐3, Par3, and NuMA [53].

Research has shown that arginine methylation significantly affects protein–protein interactions. Examples such as PRMT1‐mediated methylation of arginine 887 on inner centromere protein, critical for binding with Aurora B, and the necessity of SCY1‐like pseudokinase 1 (SCYL1) methylation for its interaction with γ2‐COP, demonstrate the functional importance of arginine modifications [40, 54]. Recent studies have identified the primary adaptor interaction site for LIC2 within residues 375 and 450 [55]. Furthermore, our study found that LIC2 is methylated by PRMT1 at the arginine 397. The methylation of LIC2 at arginine 397 could have functional implications for cellular processes. This site's modification may influence how LIC2 interacts with other proteins, such as cargoes and structural elements within the cell. Understanding the impact of this methylation will require further investigation, which could shed light on the regulatory mechanisms involving LIC2 and potentially offer new targets for therapeutic intervention in methylation‐related diseases.

Author Contributions

Jie Ran and Te Li designed the experiment and wrote the manuscript. Weiwen Bu, Jie Di, Junkui Zhao, Ruming Liu, and Yue Wu performed experiments and analyzed data.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Appendix S1

JCLA-38-e25030-s001.docx (858.8KB, docx)

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (32100614).

Funding: This work was supported by the National Natural Science Foundation of China (32100614).

Contributor Information

Jie Ran, Email: jran@sdnu.edu.cn.

Te Li, Email: 2120160955@mail.nankai.edu.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1

JCLA-38-e25030-s001.docx (858.8KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Journal of Clinical Laboratory Analysis are provided here courtesy of Wiley

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