ctdspl2b (CTD small phosphatase-like 2b) is one of two zebrafish co-orthologs of vertebrate CTDSPL2 (SCP4), a nuclear protein phosphatase of the haloacid dehalogenase (HAD) superfamily; its paralog is ctdspl2a. The protein has a disordered N-terminal regulatory region, which in the human protein carries nuclear localization sequences, and a C-terminal FCP1-homology phosphatase domain with the magnesium-dependent DxDx(T/V) catalytic motif. Human and mouse CTDSPL2 dephosphorylate serine and threonine residues on several nuclear substrates, including the RNA polymerase II C-terminal domain (Ser5-preferential), FoxO1/FoxO3a (promoting their nuclear retention and target-gene transcription, including gluconeogenic genes), Smad1/5/8 (attenuating BMP signalling) and mitotic histone H3 Thr3. The zebrafish protein keeps the catalytic motif (DLDET) and a phosphatase domain 96.2% identical to human CTDSPL2, and is 70.9% identical to ctdspl2a overall. ctdspl2b is maternally supplied and expressed throughout development and in all adult tissues sampled, in the same pattern as ctdspl2a but at two- to three-fold lower levels. No functional study of the zebrafish gene has been published.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004721 phosphoprotein phosphatase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phosphoprotein phosphatase activity, propagated from the Dullard/SCP family node PTN000258719, whose experimental sources include human CTDSPL2 itself (Q05D32). Reason: The function is ancestral to the family (FCP/SCP, Dullard/CTDNEP1, TIMM50 and plant and yeast homologs are all protein phosphatases), and ctdspl2b keeps every feature needed for it. The catalytic DxDx(T/V) motif is present as DLDET, aligned exactly to human D293-T297, and the FCP1-homology domain is 96.2% identical to human CTDSPL2. Mutating either aspartate of that motif abolishes human SCP4 function. This is the core molecular function of the gene; a more specific child (protein serine/threonine phosphatase activity) is recorded in core_functions. Supporting Evidence: PMID:35021089 SCP4 belongs to the haloacid dehalogenase (HAD) family of proteins, which employ conserved active site aspartates within a DxDx(V/T) motif to remove phosphates from protein or non-protein substrates PMID:35021089 Despite similar expression levels to the wild-type protein, SCP4D293A, SCP4D295A, and SCP4D293A/D295A mutants were unable to support MOLM-13 growth (Figures 3G and 3H). file:DANRE/ctdspl2a/ctdspl2a-bioinformatics/RESULTS.md Both copies keep the DxDx(T/V) catalytic motif (DLDET) and a phosphatase domain at least 96% identical to human CTDSPL2. |
| GO:0004721 phosphoprotein phosphatase activity | IEA GO_REF:0000117 | ACCEPT | Summary: Phosphoprotein phosphatase activity from an ARBA machine-learning rule. Reason: Same assertion as the IBA row, consistent with the conserved catalytic motif and phosphatase domain. Duplicate rows with different evidence are acceptable. Supporting Evidence: file:DANRE/ctdspl2a/ctdspl2a-bioinformatics/RESULTS.md Both copies keep the DxDx(T/V) catalytic motif (DLDET) and a phosphatase domain at least 96% identical to human CTDSPL2. |
| GO:0005634 nucleus | IEA GO_REF:0000117 | ACCEPT | Summary: Nucleus from an ARBA rule. Reason: Human and mouse CTDSPL2 are nuclear, chromatin-associated phosphatases, and their N-terminal segment carries importin-binding nuclear localization sequences. Its best-characterized substrates (Pol II CTD, FoxO1/3a, Smad1/5/8, histone H3) are nuclear proteins. The zebrafish N-terminal region is less conserved (47% identical) than the phosphatase domain, so nuclear targeting is an inference, but a well supported one for the family. Supporting Evidence: PMID:26920047 Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation. PMID:35021089 This result suggests that a function of the SCP4 N-terminal segment is to promote nuclear localization of the full-length protein. |
| GO:0016791 phosphatase activity | IEA GO_REF:0000002 | ACCEPT | Summary: Phosphatase activity from the InterPro Dullard-like phosphatase domain (IPR011948) mapping. Reason: Correct but general; the domain-level mapping cannot say more than phosphatase activity. The protein-level term (phosphoprotein phosphatase activity) is carried by the IBA row, so this IEA adds no error. Supporting Evidence: PMID:26674342 It is well known that CTDSPL2 is a nuclear protein containing FCPH domain and DXDX(T/V) motif. |
| GO:0045722 positive regulation of gluconeogenesis | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Positive regulation of gluconeogenesis from an ARBA rule, reflecting the mouse and human finding that SCP4 dephosphorylates FoxO1/3a in hepatocytes. Reason: In mammals SCP4 promotes hepatic gluconeogenesis by dephosphorylating FoxO1/3a and increasing transcription of PEPCK1 and G6PC, and SCP4 knockout mice are hypoglycemic as neonates. That is a direct role (the phosphatase acts on the transcription factors that drive the gluconeogenic genes), not just a requirement. ctdspl2b is expressed in zebrafish liver, but the role has not been examined in fish, and it is one of several physiological roles of the enzyme (FoxO in muscle and cartilage, BMP-Smad, Pol II, mitosis). Kept as a plausible, non-core, homology-based process; the same call is made for ctdspl2a, which is also expressed in liver. Supporting Evidence: PMID:28851713 In this study, we discovered a nuclear phosphatase, SCP4/CTDSPL2 (SCP4), that dephosphorylated FoxO1/3a and promoted FoxO1/3a transcription activity. PMID:28851713 Moreover, we demonstrated that gene ablation of SCP4 led to hypoglycemia in neonatal mice. |
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Download this section (compressed HTML)Q: Do ctdspl2a and ctdspl2b act on the same nuclear substrates in zebrafish (Pol II CTD, FoxO, Smad1/5/9), and is the divergent N-terminal region of either copy still able to target it to the nucleus?
Q: Is the combined ctdspl2a + ctdspl2b expression in zebrafish comparable to the single gar gene, as expected if the pair is retained for dosage?
Experiment: Generate single and double mutants (RNA-less alleles where possible, to avoid transcriptional adaptation); test early BMP-dependent dorsoventral patterning, larval glucose levels under fasting, and cartilage development; test cross-rescue by mRNA injection of each copy and of catalytic-dead (D-to-N) forms.
Hypothesis: ctdspl2a and ctdspl2b are functionally redundant phosphatases.
Type: genetic interaction
Experiment: In vitro phosphatase assays of recombinant ctdspl2a and ctdspl2b on phospho-CTD and phospho-Smad1 peptides, with human CTDSPL2 as control.
Hypothesis: Both zebrafish proteins keep CTD phosphatase activity.
Type: biochemical assay
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