SDF-9 (synthetic dauer formation 9; also EAK-5) is a 345-residue protein consisting almost entirely of a single protein tyrosine phosphatase (PTP)-like domain that has lost the catalytic machinery of the family: the nucleophilic cysteine of the HCxxGxxR(S/T) signature is replaced by serine (Ser223), and the P-loop histidine, P-loop serine/threonine and the phosphotyrosine-recognition tyrosine are also substituted. It is therefore a pseudophosphatase. SDF-9 is expressed specifically in the two XXXL/R head cells, endocrine cells that also express the dafachronic acid-synthesizing cytochrome P450 DAF-9, and it is a peripheral protein of the XXX plasma membrane whose membrane association does not require DAF-2 insulin-receptor signalling. Loss of sdf-9 causes partial, dauer-like larval arrest resembling daf-9 and daf-12 dauer-constitutive mutants, enhanced by cholesterol deprivation and suppressed by DAF-9 overexpression and by daf-16/FoxO loss. Genetically, SDF-9 acts with the membrane-associated EAK-4 and the PTP homolog EAK-6 in a single pathway, in parallel to AKT-1, to promote reproductive development, probably by potentiating DAF-9 dafachronic acid production in XXX cells. Its molecular activity is unknown; proposed roles as a phosphotyrosine-binding adaptor or a stabilizer of activated DAF-2 have not been substantiated biochemically.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004725 protein tyrosine phosphatase activity | IKR NOT PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: NOT annotation: SDF-9 lacks protein tyrosine phosphatase activity. Hu et al. note that SDF-9 lacks the catalytic cysteine found in all known PTPs and predict it to be catalytically inactive. A local alignment to PTP1B confirms the signature motif HCSAGIGRSG aligns gap-free to SDF-9 QSARGSSRAG, so the nucleophile Cys215 is Ser223 in SDF-9, and the P-loop His214 (Gln) and Ser222 (Ala) and pTyr-loop Tyr46 (Val) are also lost. No phosphatase activity has been reported for SDF-9. The NOT annotation is correct and is the appropriate record of pseudophosphatase status. Reason: Loss of the catalytic nucleophile plus several other invariant catalytic residues is the strongest sequence-level evidence of catalytic inactivity; this is the canonical PSEUDOENZYME case and the NOT annotation correctly blocks family-level propagation of GO:0004725 to SDF-9. Supporting Evidence: PMID:16839187 Whereas SDF-9 does not retain the canonical catalytic cysteine residue found in all known PTPs and is therefore predicted to be catalytically inactive file:worm/sdf-9/sdf-9-bioinformatics/RESULTS.md The catalytic cysteine position is **Ser223** in the current SDF-9 sequence. |
| GO:0005737 cytoplasm | EXP PMID:12783794 SDF-9, a protein tyrosine phosphatase-like molecule, regulat... | KEEP AS NON CORE | Summary: UniProt records cytoplasmic localization (with a dendrite-like structure in XXXL/R) from the SDF-9::GFP fusion in Ohkura et al. 2003. The cached record is abstract-only, so the localization data cannot be inspected here. The later full-text study shows SDF-9::GFP at the plasma membrane of XXX; a peripheral membrane protein without transmembrane segments is compatible with a cytoplasmic-side description. Experimental annotation from the full text is deferred to. Reason: Not contradicted, but the more informative and better documented location is the cytoplasmic face of the plasma membrane (PMID:16839187); cytoplasm is retained as non-core. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic mapping from the UniProt subcellular-location keyword (Cytoplasm), itself derived from the Ohkura et al. 2003 experimental observation. Redundant with the EXP annotation. Reason: Consistent with experimental data but redundant and less informative than plasma membrane. |
| GO:0005886 plasma membrane | IDA PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: Full-length SDF-9::GFP localizes to the plasma membrane of the XXX cells, and this localization persists in daf-2(e1370) mutants. SDF-9 has no transmembrane segment or myristoylation motif, so it is a peripheral membrane protein. Reason: Direct imaging of a functional fusion protein in the native cells. Supporting Evidence: PMID:16839187 EAK-4::GFP, SDF-9::GFP, and EAK-6::GFP fusion proteins localize to the plasma membrane of XXX. PMID:16839187 SDF-9::GFP exhibited plasma membrane localization in both wild-type and daf-2(e1370) animals |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from the UniProt Cell membrane subcellular-location annotation, which is based on PMID:16839187. Consistent with the IDA annotation. Reason: Correct and backed by direct experimental evidence. |
| GO:0007165 signal transduction | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Broad PAINT inference from a deep PTP node (PTN008567661). Unlike catalytic activity, participation in signal transduction does not depend on the catalytic cysteine, and for SDF-9 it is independently supported by genetics placing it in DAF-2 insulin-like signalling (daf-16 suppression; acts in parallel to AKT-1). The term is true but very general. Reason: Not a pseudoenzyme over-propagation (the process does not require catalysis), and consistent with sdf-9 genetics, but too general to represent the core role; the specific pathway role is captured by dauer larval development. Supporting Evidence: PMID:16839187 This supports the contention that EAK-4, SDF-9, and EAK-6 are components of DAF-2/InsR signaling. |
| GO:0040024 dauer larval development | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning inference, consistent with the experimental IMP/IGI annotations from the same gene. Reason: Correct; redundant with experimental annotations. |
| GO:0040024 dauer larval development | IGI PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: Genetic interaction (enhancer/epistasis) data from the eak screen: sdf-9 mutations strongly enhance akt-1 null dauer arrest, and sdf-9 phenotypes are suppressed by daf-16 and epistatic to daf-18/akt-1 gain-of-function alleles. Reason: Well-documented genetic interaction in full text. Supporting Evidence: PMID:16839187 eak-4, sdf-9, and eak-6 mutants strongly enhance the akt-1 null phenotype (Figure 1A), suggesting that EAK proteins function in parallel to AKT-1. |
| GO:0040024 dauer larval development | IGI PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: Genetic interaction data from the eak screen (enhancer/suppressor and double-mutant analysis with DAF-2 pathway genes) supporting a role of sdf-9 in the dauer decision. Reason: Experimental genetic evidence in full text supports the process. Supporting Evidence: PMID:16839187 implicate EAK-4, SDF-9, and EAK-6 function upstream of or in parallel to AGE-1/PI3K and AKT-1 |
| GO:0040024 dauer larval development | IGI PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: Genetic interaction data: sdf-9 dauer arrest is suppressed by daf-16/FoxO loss of function, as for all known DAF-2 pathway components. Reason: Experimental epistasis supports involvement in dauer development. Supporting Evidence: PMID:16839187 eak-4, sdf-9, and eak-6 phenotypes are fully suppressed by mutations in daf-16/FoxO |
| GO:0040024 dauer larval development | IGI PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: Genetic interaction data from the eak double-mutant and epistasis analyses supporting a role for sdf-9 in the dauer decision. Reason: Experimental genetic evidence in full text supports the process. Supporting Evidence: PMID:16839187 indicating that EAK-4, SDF-9, and EAK-6 function in the same pathway or complex |
| GO:0040024 dauer larval development | IMP PMID:16839187 Two membrane-associated tyrosine phosphatase homologs potent... | ACCEPT | Summary: sdf-9 loss-of-function alleles cause dauer arrest (weak at 25-27 C in Hu et al.; stronger in the Ohkura et al. background), forming partial dauers with cuticular alae but without pharyngeal remodelling. SDF-9 acts to promote reproductive development, i.e. it negatively regulates dauer entry, in the XXX endocrine cells. Reason: Core biological role, directly supported by mutant phenotypes in two independent studies. The WormBase convention uses dauer larval development for Daf-c genes; a regulation term (negative regulation of dauer larval development) would be more precise but the existing term is acceptable. Supporting Evidence: PMID:16839187 we have observed that eak-4, sdf-9, and eak-6 single mutants form partial dauers that have cuticular alae but have not undergone pharyngeal remodeling PMID:12783794 The dauer larva of sdf-9 mutants is different from a normal dauer larva but resembles the dauer-like larva of daf-9 and daf-12 dauer-constitutive mutants. |
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Download this section (compressed HTML)Q: UniProt's CAUTION states the catalytic cysteine is replaced by lysine at position 223, but the current G5EGA9 sequence has serine at 223 (QSARGSSRAG aligned to PTP1B HCSAGIGRSG). Should the CAUTION text be corrected?
Q: Does SDF-9 directly bind DAF-2 (as proposed from allele-specific genetics) or tyrosine-phosphorylated proteins in vivo, given that heterologous co-IP in 293T cells was negative?
Q: Would positive regulation of insulin receptor signaling pathway (GO:0046628) or negative regulation of dauer larval development (GO:0061067) be a more precise process annotation for sdf-9 than dauer larval development, given genetic placement upstream of or in parallel to AGE-1/AKT-1 rather than demonstrated participation?
Q: Does SDF-9 act as the non-catalytic partner of EAK-6 (same PANTHER subfamily SF561), by analogy with the inactive D2 domains of receptor PTPs?
Experiment: Restore the catalytic cysteine (S223C, with Q222H and A230S) in an sdf-9 rescue construct and test whether a catalytically competent version changes rescue of the dauer phenotype; in parallel, assay recombinant SDF-9 and the restored mutant for PTP activity against pNPP and phosphopeptides.
Experiment: Endogenous tagging of SDF-9 followed by XXX-cell-specific proximity labelling (TurboID) to identify membrane partners (e.g. DAF-2, EAK-4, EAK-6) and test whether SDF-9 controls DAF-9 levels or dafachronic acid output (rescue of sdf-9 Daf-c by exogenous dafachronic acid).
sdf-9 encodes a protein tyrosine phosphatase, paralogous to EAK-6; sdf-9 acts in parallel to akt-1 to regulate insulin-like signaling and dauer formation and may promote DAF-9 activity by means of steroid hormone signalling; sdf-9 dauer larvae resemble those of daf-9 and daf-12 dauer-constitutive mutants, and these Daf-c mutants are all enhanced by cholesterol deprivation; SDF-9 is expressed in the neuroendocrine XXXL/R cells, where it associates with the plasma membrane; ablation of the XXXL/R cells in wild-type L1 larvae also causes daf-9-like pseudodauer larvae.
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