SID-3 is a cytoplasmic non-receptor tyrosine kinase of the ACK (activated Cdc42-associated kinase) family. It has an N-terminal tyrosine kinase domain, an SH3 domain and a Cdc42/Rac-interactive binding (CRIB) domain, and is the C. elegans ortholog of human ACK1/TNK2. SID-3 is expressed broadly (intestine, pharynx, body wall muscle, epidermis, excretory canal) and localizes diffusely and in puncta in the cytoplasm. It acts in recipient cells to promote efficient import of double-stranded RNA and mobile silencing RNA, and so is needed for systemic RNA interference. It is not required for cell-autonomous RNAi. This import role needs an intact kinase domain (the K139A ATP-binding mutant does not rescue) and has been proposed to work through endocytosis. SID-3 kinase activity is also required for infection by the Orsay virus at an early, pre-replication step, probably entry. In addition, sid-3 mutants constitutively derepress a set of antiviral response genes that are bound and repressed by the STAT factor STA-1, which places SID-3 genetically upstream of STA-1-mediated repression. The substrates of SID-3 are unknown.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based assignment of protein kinase activity from the kinase domain. Reason: Correct but general. SID-3 has a conserved tyrosine kinase domain whose catalytic lysine (K139) is required for function. The more specific tyrosine kinase terms capture this activity better. Supporting Evidence: PMID:22912399 Thus, the kinase activity of SID-3 is essential to enable efficient import of dsRNA into C. elegans cells. |
| GO:0004713 protein tyrosine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA transfer of protein tyrosine kinase activity across the non-receptor tyrosine kinase family. Reason: SID-3 is an ACK-family tyrosine kinase, inside the clade covered by this PAINT node, with no sign of catalytic loss. A kinase-dead K139A mutant fails to rescue either dsRNA import or Orsay virus infection. Supporting Evidence: PMID:22912399 This domain composition is present in the activated Cdc-42–associated kinase (Ack) family of cytoplasmic tyrosine kinases PMID:28874467 Our experiments suggest that the kinase activity of SID-3 is important for its ability to promote Orsay virus infection. |
| GO:0004713 protein tyrosine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA assignment of protein tyrosine kinase activity (InterPro IPR020635, Rhea 10596). Reason: Consistent with the tyrosine kinase domain and with the genetic requirement for kinase activity. Supporting Evidence: PMID:22912399 Thus, the kinase activity of SID-3 is essential to enable efficient import of dsRNA into C. elegans cells. |
| GO:0004715 non-membrane spanning protein tyrosine kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.7.10.2 mapping to non-membrane spanning protein tyrosine kinase activity. Reason: The most precise MF term for SID-3. ACK kinases are cytoplasmic (non-receptor) tyrosine kinases, and SID-3 has no transmembrane segment and is cytoplasmic. This is the core activity. Supporting Evidence: PMID:28874467 sid-3 encodes a nonreceptor tyrosine kinase that was originally identified in a genetic screen for animals defective in systemic RNAi |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based ATP binding from the protein kinase ATP-binding site. Reason: K139 corresponds to the ATP-binding lysine of ACK1 (K158), and mutating it abolishes SID-3 function. ATP binding is part of the kinase mechanism, not a separate core function. Supporting Evidence: PMID:22912399 This lysine residue corresponds to ACK1(K158), which is required for ATP binding |
| GO:0005737 cytoplasm | IDA PMID:22912399 Conserved tyrosine kinase promotes the import of silencing R... | ACCEPT | Summary: Rescuing SID-3::DsRed and SID-3::GFP fusions are diffuse and punctate in the cytoplasm of all tissues examined. Reason: Direct localization with functional (rescuing) fusion proteins. Supporting Evidence: PMID:22912399 This fusion protein was also broadly expressed and localized to the cytoplasm of cells in a diffuse as well as punctate pattern |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to cytoplasm. Reason: Matches the experimental localization. Supporting Evidence: PMID:22912399 These results suggest that the SID-3 protein likely functions in the cytoplasm of most tissues to enable efficient RNAi in response to ingested dsRNA. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA transfer of plasma membrane localization from a broad non-receptor tyrosine kinase node (Src, Tec, Syk, Abl, FAK families). Reason: Many members of this node act at the plasma membrane. ACK-family kinases are recruited to membranes and endocytic vesicles by Cdc42 and receptors, so transient membrane association of SID-3 is plausible. Experimentally, SID-3 has only been seen as diffuse and punctate cytoplasmic signal. Kept as non-core, not as a defining location. Supporting Evidence: PMID:22912399 Third, in cultured mammalian cells, activated ACK associates with Cdc42 and invaginated endocytic vesicles |
| GO:0035194 regulatory ncRNA-mediated post-transcriptional gene silencing | IMP PMID:22912399 Conserved tyrosine kinase promotes the import of silencing R... | MODIFY | Summary: sid-3 mutants are defective in silencing by mobile and ingested dsRNA in many tissues, but are not defective (and are marginally enhanced) in cell-autonomous RNAi. Reason: The phenotype is real, but the full text shows that SID-3 is not part of the silencing machinery. sid-3 mutants execute RNAi normally when dsRNA is injected into the syncytial germline. Tissue-specific rescue shows SID-3 works in the recipient cell to import dsRNA and mobile silencing RNA. The function SID-3 performs is therefore dsRNA import into cells, upstream of silencing. GO:0033227 dsRNA transport describes the step SID-3 contributes to more accurately than the general gene-silencing process. Proposed replacements: dsRNA transport Supporting Evidence: PMID:22912399 Nevertheless, these results clearly demonstrate that sid-3 mutants are not defective in the execution of RNAi. PMID:22912399 Thus, these results support the idea that SID-3 is specifically required to ensure the efficient import of mobile silencing RNA and dsRNA into C. elegans cells. |
| GO:0050687 negative regulation of defense response to virus | IMP PMID:28874466 An Alternative STAT Signaling Pathway Acts in Viral Immunity... | NEW | Summary: sid-3 mutants constitutively upregulate antiviral response genes that overlap strongly with genes derepressed in sta-1 mutants and are enriched for STA-1 ChIP-seq binding. Reason: The RNA-seq data place the SID-3 kinase genetically upstream of STA-1-mediated repression of antiviral genes. SID-3 is a signalling kinase in this pathway, not a substrate, so a regulatory process term applies. This repression role is separate from the SID-3 requirement for virus entry: sta-1;sid-3 double mutants remain resistant. Evidence is transcriptomic and genetic, and the direct SID-3 substrate (possibly STA-1) is unknown. Supporting Evidence: PMID:28874466 Moreover, genes upregulated in sid-3 mutants, including those shared with sta-1, were enriched for STA-1 binding by ChIP-seq, suggesting that sid-3 acts upstream of sta-1 in the antiviral gene expression response |
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Download this section (compressed HTML)Q: What are the direct substrates of SID-3? Does SID-3 phosphorylate STA-1, or the dynamin/clathrin endocytic machinery used for dsRNA and virus uptake?
Q: Is Cdc42 binding through the CRIB domain needed for SID-3 activation in dsRNA import and in Orsay virus entry?
Experiment: Run phosphotyrosine proteomics on wild-type, sid-3 null and kinase-dead K139A animals to identify SID-3 substrates. Test STA-1 tyrosine phosphorylation directly.
Experiment: Test whether CRIB-domain point mutants of SID-3 rescue systemic RNAi and Orsay virus susceptibility in sid-3 mutants.
sid-3 encodes a nonreceptor tyrosine kinase that is a member of the activated cdc-42-associated kinase (Ack) subfamily of cytoplasmic tyrosine kinases; in C. elegans sid-3 functions to regulate the import of dsRNA into cells, thus affecting the efficiency of RNA-mediated gene silencing (RNAi); SID-3 localizes to the cytoplasm.
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