CLK4

CLK4 is a dual-specificity protein kinase that governs alternative splicing through phosphorylation of serine/arginine-rich (SR) splicing factors and also acts as a signaling node controlling transcription factor turnover and stress-responsive pathways (PMID:11170754, PMID:40126184). Catalytically, it autophosphorylates and phosphorylates substrates such as myelin basic protein, with kinase activity strictly dependent on the K189 residue (PMID:11170754, PMID:35092699). Its principal cellular function is the phosphorylation of SR proteins including SRSF4 and SRSF6; selective inhibition of CLK4 reduces SR-protein phosphorylation, alters their nuclear distribution, and produces widespread changes in alternative splicing and RNA processing, coupled to G2/M arrest and apoptosis (PMID:38009092, PMID:40126184). Beyond splicing, active CLK4 phosphorylates the transcription factor MITF at Y360, promoting its interaction with the E3 ligase COP1, K63-linked ubiquitination, and sequestosome-1-mediated autophagic degradation, a modification reversed by the deubiquitinase ZRANB1 (PMID:35092699). CLK4 also functions as an essential relay in DNA damage-induced NF-κB activation, transmitting signals between ATM/PARP1 and IKKγ, and it mediates TGF-β-driven EMT, invasion, and cancer stem cell properties in triple-negative breast cancer (PMID:37506701, PMID:35046528). Its catalytic output is itself redox-tunable: methionine oxidation at M307 impairs kinase activity (PMID:35092699). The SR-related protein Clasp binds specifically the kinase-dead form of CLK4, linking inactive kinase to regulation of Clk-family alternative splicing (PMID:12169693).

1. 2001 Medium

   Established that human CLK4 is an enzymatically active dual-specificity kinase, defining its basic catalytic capacity and substrate selectivity.

   Evidence GST-fusion in vitro kinase assay with myelin basic protein and histone H2B as candidate substrates

   PMID:11170754

   Open questions at the time

   • No physiological substrate identified
   • Single method, no mutagenesis validation
   • Catalytic residues not mapped
2. 2002 Medium

   Identified Clasp as a partner that selectively engages the inactive (kinase-dead) form of CLK4, suggesting kinase state controls splicing-regulatory complex assembly.

   Evidence Yeast two-hybrid screen plus in vitro binding and an overexpression alternative splicing readout on Clk1 pre-mRNA

   PMID:12169693

   Open questions at the time

   • Mechanism of state-selective binding unknown
   • Functional consequence shown only on overexpression
   • Single lab
3. 2022 High

   Defined a non-splicing substrate axis in which active CLK4 phosphorylates MITF at Y360 to drive its ubiquitin- and autophagy-dependent degradation, with redox modification tuning kinase output.

   Evidence In vitro kinase assay, Co-IP, GST pulldown, K189R kinase-dead mutant, ubiquitination assays, plus mass spectrometry of M307 oxidation with metabolic readouts in ESCC cells

   PMID:35092699

   Open questions at the time

   • Single lab
   • Physiological trigger of M307 oxidation undefined
   • Direct kinase-substrate contact at Y360 not structurally resolved
4. 2022 Medium

   Placed CLK4 functionally within TGF-β-driven metastatic programs, linking its activity to EMT and invasion in TNBC.

   Evidence RNAi knockdown, in vitro invasion assays, mouse xenograft, and pharmacological CLK4 inhibition

   PMID:35046528

   Open questions at the time

   • Direct CLK4 substrates in the TGF-β pathway not identified
   • Mechanistic link between kinase activity and EMT gene expression unresolved
5. 2023 Medium

   Positioned CLK2/CLK4 as essential transducers in DNA damage-induced NF-κB activation, identifying a signaling step between ATM/PARP1 and IKKγ.

   Evidence Multi-kinase panel deconvolution, inhibitor derivatization, in vitro kinase inhibition, and NF-κB activation assays under DNA damage

   PMID:37506701

   Open questions at the time

   • Direct CLK4 substrate in the NF-κB relay not identified
   • CLK4 contribution not fully separated from CLK2
6. 2023 Medium

   Used a selective chemical probe to causally link CLK4 (with CLK1/2) activity to SR-protein phosphorylation status and nuclear localization.

   Evidence Selective inhibitor (SGC-CLK-1/CAF-170), phosphorylation assays, subcellular localization imaging, reversibility tests

   PMID:38009092

   Open questions at the time

   • CLK4 effects not separated from CLK1/2
   • Specific SR substrates of CLK4 not delineated here
7. 2025 Medium

   Demonstrated with a CLK4-selective inhibitor that CLK4 phosphorylates SRSF4/SRSF6 to control alternative splicing, coupling its kinase activity to cell cycle and survival outcomes.

   Evidence Selective inhibitor (compound 150441), in vitro kinase and phosphorylation assays, cell cycle and viability analysis, RNA-seq in pancreatic cancer cells

   PMID:40126184

   Open questions at the time

   • Direct phosphosites on SRSF4/SRSF6 not mapped
   • Single lab
   • Causal splicing events driving phenotype not isolated

• How CLK4's distinct functions — SR-protein-directed splicing, MITF degradation, TGF-β/EMT, and DNA-damage NF-κB signaling — are coordinated, and whether common direct substrates or regulatory inputs unify them, remains unresolved.
• No structural model of substrate engagement
• Substrate repertoire incompletely defined
• Cross-talk between splicing and signaling roles unknown