CDKAL1 is a radical S-adenosyl-L-methionine (SAM) methylthiotransferase of the MiaB family (e-MtaB subfamily). It converts N6-threonylcarbamoyladenosine at position 37 (t6A37) of cytosolic tRNA-Lys(UUU) into 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37), using two [4Fe-4S] clusters, one of which binds SAM and generates the 5'-deoxyadenosyl radical. The protein has an N-terminal MTTase domain, a central radical SAM domain and a TRAM domain. A C-terminal transmembrane segment makes it a tail-anchored protein of the endoplasmic reticulum membrane, with the catalytic domains facing the cytosol. The ms2t6A37 modification stabilizes codon-anticodon pairing and prevents misreading of the lysine codons AAA and AAG. In pancreatic beta cells, loss of CDKAL1 causes lysine misincorporation into proinsulin, defective proinsulin processing, ER stress and reduced first-phase glucose-stimulated insulin secretion. This explains the association of common CDKAL1 variants with type 2 diabetes. Despite its name, CDKAL1 does not bind the CDK5 activator p35 or inhibit CDK5.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM signatures (IPR006638, IPR007197) to the root catalytic activity term. Reason: True but uninformative. Per the RADICAL_SAM treatment rule, catalytic activity is replaced by the specific reaction CDKAL1 carries: t6A37 tRNA methylthiotransferase (EC 2.8.4.5), shown in E. coli complementation (PMID:20584901) and in Cdkal1 knockout mice (PMID:21841312), and already carried by this gene as GO:0035598. Proposed replacements: tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity Supporting Evidence: PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | REMOVE | Summary: Yeast two-hybrid interaction with LCK from the HuRI proteome-scale interactome map. Reason: Bare protein binding from a high-throughput interaction screen. It records no molecular function, and no follow-up links the interaction to CDKAL1 tRNA methylthiotransferase activity. Removed under the protein-binding policy; this does not assert the interaction is false. Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Q5VV42; P06239: LCK; NbExp=3; IntAct=EBI-10194801, EBI-1348; |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: Interaction with WFS1 from a neurodegenerative-disease protein interactome screen. Reason: Bare protein binding from a high-throughput interaction screen. It records no molecular function, and no follow-up links the interaction to CDKAL1 tRNA methylthiotransferase activity. Removed under the protein-binding policy; this does not assert the interaction is false. Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Q5VV42; O76024: WFS1; NbExp=3; IntAct=EBI-10194801, EBI-720609; |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: Interaction with ATXN3 from a neurodegenerative-disease protein interactome screen. Reason: Bare protein binding from a high-throughput interaction screen. It records no molecular function, and no follow-up links the interaction to CDKAL1 tRNA methylthiotransferase activity. Removed under the protein-binding policy; this does not assert the interaction is false. Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Q5VV42; P54252: ATXN3; NbExp=3; IntAct=EBI-10194801, EBI-946046; |
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference of ER localization for the vertebrate CDKAL1 clade, with mouse Cdkal1 and human CDKAL1 among the sources. Reason: CDKAL1 is in the endoplasmic reticulum, but more precisely it is a tail-anchored integral protein of the ER membrane, with its catalytic domain on the cytosolic face (PMID:23048041; Reactome). The gene already carries GO:0005789 endoplasmic reticulum membrane (EXP), so the general term is refined to it. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000228711 SUPPORTS TRANSFER ER localization of the vertebrate CDKAL1 clade is sound (mouse Cdkal1 and human CDKAL1 are experimental sources); only the granularity is refined to ER membrane. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: PMID:23048041 We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. PMID:21841312 This hydrophobic domain was determined to carry the ER-localization signal because deletion of this domain disrupted ER localization (Figure 2 D). |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000107 | MODIFY | Summary: Ensembl Compara projection of ER localization from mouse Cdkal1 (Q91WE6). Reason: CDKAL1 is in the endoplasmic reticulum, but more precisely it is a tail-anchored integral protein of the ER membrane, with its catalytic domain on the cytosolic face (PMID:23048041; Reactome). The gene already carries GO:0005789 endoplasmic reticulum membrane (EXP), so the general term is refined to it. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: PMID:23048041 we further established that CDKAL1 is an endoplasmic reticulum-resident protein. PMID:21841312 This hydrophobic domain was determined to carry the ER-localization signal because deletion of this domain disrupted ER localization (Figure 2 D). |
| GO:0005789 endoplasmic reticulum membrane | EXP PMID:23048041 CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1)... | ACCEPT | Summary: Brambillasca et al. 2012 showed that CDKAL1 is a tail-anchored protein inserted into the ER membrane through the TRC40/Get3 pathway, with ER residence confirmed by glycosylation-reporter assays in insulinoma cells. Reason: Direct experimental evidence for the site of action. The C-terminal transmembrane anchor (UniProt TRANSMEM 556-578) holds the enzyme on the cytosolic face of the ER membrane, where it modifies cytosolic tRNA-Lys(UUU). Supporting Evidence: PMID:23048041 We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. PMID:23048041 we further established that CDKAL1 is an endoplasmic reticulum-resident protein. |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (SL-0097, endoplasmic reticulum membrane). Reason: Agrees with the experimental localization (PMID:23048041) on which the UniProt location is based. Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Endoplasmic reticulum membrane PMID:23048041 We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-6786571 | ACCEPT | Summary: Reactome reaction for CDKAL1:4Fe-4S methylthiolating t6A37, placing CDKAL1 on the cytosolic face of the ER. Reason: Agrees with the experimental data (PMID:23048041). Supporting Evidence: Reactome:R-HSA-6786571 CDKAL1 is located on the cytosolic face of the endoplasmic reticulum therefore the reaction is presumed to occur in the cytosol |
| GO:0005791 rough endoplasmic reticulum | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ensembl Compara projection of rough ER localization from mouse Cdkal1. Reason: Supported by subcellular fractionation of mouse liver in Wei et al. 2011. This is a sub-compartment observation from fractionation that adds little to the ER-membrane localization, so it is kept as non-core. Supporting Evidence: PMID:21841312 Furthermore, endogenous Cdkal1 was detected in the rough ER fraction purified from mouse liver (Supplemental Figure 6). |
| GO:0006400 tRNA modification | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the methylthiotransferase family signature (IPR005839) to tRNA modification. Reason: True but general. CDKAL1 performs one specific tRNA modification, 2-methylthiolation of t6A37, so the process is refined to GO:0035600 tRNA methylthiolation, which the gene already carries. Proposed replacements: tRNA methylthiolation Supporting Evidence: PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). |
| GO:0008150 biological_process | ND GO_REF:0000015 | MODIFY | Summary: No-data placeholder for biological process. Reason: The placeholder has been overtaken by experimental data: CDKAL1 performs tRNA methylthiolation of t6A37 (PMID:20584901, PMID:21841312). The root term is replaced by that process. Proposed replacements: tRNA methylthiolation Supporting Evidence: PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | MODIFY | Summary: Detected in a mass-spectrometry membrane proteome of the NK-like cell line YTS. Reason: Correct but general. The membrane in question is the ER membrane, into which CDKAL1 is inserted as a tail-anchored protein (PMID:23048041); the term is refined to GO:0005789, which the gene already carries. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: PMID:19946888 Mass spectrometric analysis identified 1843 proteins with high confidence scores. PMID:23048041 We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. |
| GO:0016740 transferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the methylthiotransferase family signature to transferase activity. Reason: True but general. The specific transferase activity is t6A37 tRNA methylthiotransferase activity (EC 2.8.4.5), which the gene already carries as GO:0035598. Proposed replacements: tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity Supporting Evidence: PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). |
| GO:0035596 methylthiotransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the MTTase N-terminal domain (IPR013848) to methylthiotransferase activity. Reason: True but general. CDKAL1 is an e-MtaB family MTTase whose substrate is t6A37 in tRNA, so the activity is refined to GO:0035598, which the gene already carries. Proposed replacements: tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Belongs to the methylthiotransferase family. CDKAL1 subfamily. PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. |
| GO:0035598 tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of t6A37 methylthiotransferase activity for the e-MtaB/CDKAL1 clade (node PTN000228764), with mouse Cdkal1 and B. subtilis MtaB (YqeV, P54462) among the experimental sources. Reason: Core function. The node is well grounded: human CDKAL1 makes ms2t6A in E. coli complementation (PMID:20584901), mouse Cdkal1 knockouts lose ms2t6A in tRNA-Lys(UUU) (PMID:21841312), and the bacterial MtaB ortholog has the same activity. Supporting Evidence: PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). PMID:21841312 The ms 2 t 6 A modification was detected only in the WT mice but not in the Cdkal1 –/– mice (Figure 1 C). |
| GO:0035598 tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (Compara from mouse Q91WE6, InterPro IPR006466, RHEA:37075, EC 2.8.4.5). Reason: Agrees with the experimental evidence for the activity (PMID:20584901, PMID:21841312). Supporting Evidence: PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). |
| GO:0035598 tRNA (N(6)-L-threonylcarbamoyladenosine(37)-C(2))-methylthiotransferase activity | ISS GO_REF:0000024 | ACCEPT | Summary: UniProt curator transfer of the activity from mouse Cdkal1 (Q91WE6), whose knockout abolishes ms2t6A in tRNA-Lys(UUU). Reason: Sound transfer between one-to-one orthologs; the human protein is also active in E. coli complementation (PMID:20584901). Supporting Evidence: PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). PMID:20584901 These results demonstrate that B. subtilis YqeV/MtaB and human CDKAL1/e-MtaB proteins are both functional in vivo and selectively involved in the conversion of t 6 A to ms 2 t 6 A. |
| GO:0035600 tRNA methylthiolation | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from IPR006466 (MiaB-like, archaeal/eukaryotic) to tRNA methylthiolation. Reason: Core biological process: CDKAL1 itself catalyses the methylthiolation step converting t6A37 to ms2t6A37 in tRNA-Lys(UUU). Supporting Evidence: PMID:21841312 These results demonstrate that mouse Cdkal1 is a methylthiolase that converts t 6 A to ms 2 t 6 A in tRNA Lys (UUU). PMID:21841312 The ms 2 t 6 A modification was detected only in the WT mice but not in the Cdkal1 –/– mice (Figure 1 C). |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM signatures to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to its descendant GO:0051539 4 iron, 4 sulfur cluster binding. CDKAL1 binds two [4Fe-4S] clusters (MTTase N-terminal cluster and radical SAM cluster), and the gene already carries GO:0051539. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:human/CDKAL1/CDKAL1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:20584901 we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the MTTase and radical SAM signatures to 4Fe-4S cluster binding. Reason: Core cofactor binding per the RADICAL_SAM treatment rule. The CxxxCxxC radical SAM cluster is required for ms2t6A formation by CDKAL1 (Cys-to-Ala mutant inactive), and the family binds two [4Fe-4S] clusters. Supporting Evidence: PMID:20584901 This provides, as expected, strong evidence that the cluster is required for activity. PMID:20584901 we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. file:human/CDKAL1/CDKAL1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:1990145 maintenance of translational fidelity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference that CDKAL1-clade enzymes act in maintenance of translational fidelity (node PTN000228764, with mouse Cdkal1 as a source). Reason: The ms2t6A37 modification that CDKAL1 installs is needed for accurate decoding of Lys AAA/AAG codons; without it Lys is misincorporated into proinsulin (PMID:21841312). The term describes the purpose of the modification, not the catalytic step, which is GO:0035600. Kept as non-core in deference to the PAINT node. Supporting Evidence: PMID:21841312 These results suggest that the 2-methylthio modification ms 2 t 6 A in tRNA Lys (UUU) is important for preventing the misreading of its cognate codons, especially when the rate of translation is relatively high. PMID:31941883 Cdkal1 catalyzes the methylthiolation of N6-threonylcarbamoyl adenosine 37 (t6A37) in cytosolic tRNALysUUU to generate ms2t6A37, which is required for the accurate translation of lysine codons in proinsulin19,20. |
| GO:1904047 S-adenosyl-L-methionine binding | ISS PMID:23542644 Two Fe-S clusters catalyze sulfur insertion by radical-SAM m... | NEW | Summary: CDKAL1 is a radical SAM methylthiotransferase. SAM binds the unique iron of the radical SAM [4Fe-4S] cluster and is reductively cleaved to the 5'-deoxyadenosyl radical; a second SAM supplies the methyl group of the methylthio moiety. Reason: Not in GOA. Added for consistency with the RADICAL_SAM treatment rule (SAM binding is a core cosubstrate-binding function of every active radical SAM enzyme) and with the LIAS and ELP3 reviews. This is a molecular function, so CDKAL1 itself must bind SAM, and it does: the reaction it catalyses (RHEA:37075) consumes two SAM, and UniProt (PROSITE-ProRule PRU00780) records an exchangeable SAM on the radical SAM cluster. The structure and mechanism of the MTTase family member RimO show SAM bound to the radical SAM cluster in the canonical geometry (PMID:23542644), and that paper names CDKAL1 as a family member. Transferred by sequence similarity (ISS) from the family. Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: PMID:23542644 Like many enzymes catalyzing biological sulfur-insertion reactions1, MiaB, CDKAL1, and RimO are iron-sulfur-containing Radical-SAM enzymes. PMID:23542644 The Radical-SAM cluster catalyzes the reductive cleavage of S-Adenosylmethionine (SAM) into the 5′-deoxyadenosyl radical (Ado•) that activates the substrate for transformation by abstracting a hydrogen atom from a specific C-H bond4. file:human/CDKAL1/CDKAL1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
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Download this section (compressed HTML)Q: Is CDKAL1's contribution to insulin secretion and glucose homeostasis entirely explained by tRNA-Lys(UUU) decoding fidelity (proinsulin and other Lys-rich beta-cell proteins), or does ER-membrane anchoring give it an additional local role near the translocon? Process terms such as insulin secretion should stay off CDKAL1 unless such a direct role is shown.
Q: Which protein donates sulfur to CDKAL1 in the cytosol (the second, N-terminal [4Fe-4S] cluster in MiaB-family enzymes is proposed to be the sulfur source), and does that require regeneration by the cytosolic Fe-S assembly (CIA) machinery?
Experiment: Reconstitute purified human CDKAL1 (soluble construct lacking the tail anchor) with [4Fe-4S] clusters and test methylthiolation of t6A37-containing tRNA-Lys(UUU) transcripts in vitro, with SAM and reductant. Measure kinetics and test whether the two clusters have separate roles.
Experiment: Ribosome profiling and mass-spectrometric measurement of Lys misincorporation across the proteome in CDKAL1-knockout human beta-cell lines (e.g. EndoC-betaH1). This would show whether proinsulin is uniquely sensitive or one of many Lys-codon-dependent targets.
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