Cdc2-like kinases: structure, biological function, and therapeutic targets for diseases

Abstract

The CLKs (Cdc2-like kinases) belong to the dual-specificity protein kinase family and play crucial roles in regulating transcript splicing via the phosphorylation of SR proteins (SRSF1-12), catalyzing spliceosome molecular machinery, and modulating the activities or expression of non-splicing proteins. The dysregulation of these processes is linked with various diseases, including neurodegenerative diseases, Duchenne muscular dystrophy, inflammatory diseases, viral replication, and cancer. Thus, CLKs have been considered as potential therapeutic targets, and significant efforts have been exerted to discover potent CLKs inhibitors. In particular, clinical trials aiming to assess the activities of the small molecules Lorecivivint on knee Osteoarthritis patients, and Cirtuvivint and Silmitasertib in different advanced tumors have been investigated for therapeutic usage. In this review, we comprehensively documented the structure and biological functions of CLKs in various human diseases and summarized the significance of related inhibitors in therapeutics. Our discussion highlights the most recent CLKs research, paving the way for the clinical treatment of various human diseases.

Fig. 1

Structural comparison of human CLKs. a Domain structures of human CLK1, -2, -3, and -4. b Crystal structure of human CLK1 without N-terminus domain (PDB ID: 6I5H). The protein structure backbone is visualized as a cartoon representation, while the ligand and its interacting residues are visualized as sticks. H-bonds are shown as black dashed lines. The coloring of all domains and signature sequence motifs are consistent with panel a. c Structural superposition of CLK1 (PDB ID: 6I5H, green), CLK2 (PDB ID: 6KHE, cyan), CLK3 (PDB ID: 2EU9, magenta) and CLK4 (PDB ID: 6FYV, yellow). The protein structural backbone is represented as a ribbon. d Electrostatic surface representation of CLKs. The blue and red colors represent positive and negative charges, respectively. The different pockets among these kinases were determined by the distances between side chains of conserved Val residue in the N-lobe and DFG-1 residue in the C-lobe

Fig. 2

Biology function of CLKs. CLKs participate in biological processes by modulating splicing and non-splicing functions. CLKs are activated, phosphorylated or regulated by c-Myc, AKT or 14–3–3τ, or negatively regulated by miRNAs. CLKs affect their downstream effectors by phosphorylating serine, threonine, or tyrosine residues to activate cellular splicing and non-splicing processes. Alternative splicing of certain genes is increased in response to protein phosphorylation by CLKs. Targets of CLKs include SR proteins, SPF45, and/or U1–70K or modulated RBFOX2. Consequently, different protein isoforms that function in multiple cellular processes are generated. CLKs promote cytokinesis, increase c-myc activity, and suppress fatty acid metabolism by phosphorylating downstream Aurora B, USP13, and PGC-1α. The activation of Wnt/β-catenin and Hippo signaling by increased expression of Wnt 3a or YAP further highlights the importance of CLKs in non-splicing processes. The figure was generated using Figdraw (www.figdraw.com)

Fig. 3

The direct upstream and downstream regulators of CLKs. The expression or activities of CLKs are directly regulated by the upstream proteins such as AKT, c-Myc, or miRNAs. CLKs phosphorylate or modulate their downstream targets, which have been demonstrated in publications, to participate in pivotal processes. The upstream and downstream regulators of CLK1 (a), CLK2 (b), CLK3 (c), and CLK4 (d) have been shown in the figure seperately. The figure was generated by Figdraw (www.figdraw.com)

Fig. 4

Function of CLKs and related inhibitors in human non-cancer diseases. CLKs participate in various human non-cancer diseases including neurodegenerative diseases, inflammatory diseases, Duchenne muscular dystrophy, viral replication, autophagy-associated diseases, and other diseases. CLKs mediate the pathological processes through modulating alternative splicing or changing transcriptional activities. The figure was generated by Figdraw (www.figdraw.com)

Fig. 5

Expression status and clinical outcomes associated with CLKs transcript levels in different tumor types. a RNA-seq data of CLKs expression in tumor and normal tissues were obtained from the TCGA database (https://portal.gdc.cancer.gov/). The Kruskal-Wallis test was used to assess statistical significance. b Raw counts of clinical survival information of CLKs were obtained from The TCGA dataset (https://portal.gdc.cancer.gov/). The KM survival analysis with log-rank test was also used to compare the survival differences between the above two groups. For Kaplan–Meier curves, p values, and hazard ratio (HR) with 95% confidence interval (CI) were generated by log-rank tests and univariate Cox proportional hazards regression. *, ** and *** represented p < 0.05, p < 0.01 and p < 0.001, separately. p < 0.05 was considered statistically significant

Fig. 6

The oncogenic function of CLKs and utilization of CLKs inhibitors in cancer. a–d CLKs participate in cancer development, invasion, and metastasis by altering mRNA splicing, wnt/β-catenin signaling, TGF-β signaling, or mediating cell cycle transition. CLKs regulate cancer growth or metastasis by phosphorylating or modulating their downstream regulators, for instance, SRSF5, SPF45, USP13 SMAD3, PP2A, and other proteins. CLKs inhibitors decreased cancer growth, metastasis, metabolism, and promoted apoptosis by modulating genes participating in cell cycle, EMT, metabolic pathway, and apoptosis, respectively. e Preclinical cancer research conducted to assess the therapeutic potential of CLKs-targeting compounds showed significant tumor growth inhibitory effects. The figure was generated by Figdraw(www.figdraw.com)