CIMAP3 (Pitchfork, PIFO; formerly C1orf88) is a small chordate-specific protein with two SHIPPO repeats, related to the sperm outer dense fiber protein ODF3 family. It is expressed in embryonic organizer regions such as the monociliated pit cells of the node, and in testis. Pitchfork travels on vesicles from the Golgi/trans-Golgi network and along the axoneme. It accumulates at the ciliary basal body when quiescent cells re-enter the cell cycle. There it associates with Aurora A kinase (AURKA) and promotes its activation, a step in the AURKA-HDAC6 pathway that resorbs primary cilia. Reduced Pitchfork dosage in mouse leaves cilia unretracted, the basal body undetached and centrosomes over-duplicated. The result is duplicated node cilia, left-right asymmetry defects and heart malformations. Pull-down assays also co-precipitate Pitchfork with ciliary trafficking components (RAB6, RAB8, ARL13B, KIF3A, centrin, beta- and gamma-tubulin). In mouse cells, Pitchfork and GPRASP2 form a Hedgehog-induced ciliary targeting complex needed for Smoothened entry into the primary cilium. In motile cilia, CIMAP3 is a microtubule-associated protein bound to the outer surface of axonemal doublet microtubules. A heterozygous R80K variant that blocks AURKA activation was found in individuals with laterality and cystic kidney phenotypes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | REMOVE | Summary: Pull-down association with centrin (CETN1); generic protein binding. Reason: Generic protein binding carries no functional information. The underlying association comes from TAP pull-down of endogenous proteins from HEK293T lysates; it does not distinguish direct from complex-mediated binding. Removing GO:0005515 does not dispute the reported association. No more informative centrin-binding MF is warranted from a lysate pull-down. Supporting Evidence: PMID:20643351 We precipitated endogenous proteins using immobilized mouse and human SF-TAP-tagged PIFO in ciliated and cilia-disassembling HEK293T cells PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0005515 protein binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | MODIFY | Summary: Pull-down association with the ciliary small GTPase ARL13B. Reason: Generic protein binding carries no functional information. The underlying association comes from TAP pull-down of endogenous proteins from HEK293T lysates; it does not distinguish direct from complex-mediated binding. Removing GO:0005515 does not dispute the reported association. ARL13B is a small GTPase; for consistency with the RAB6A/RAB8A rows from the same experiment the informative term is small GTPase binding (kept as non-core). Proposed replacements: small GTPase binding Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0005634 nucleus | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Nucleus, electronic transfer from mouse Pifo, which has a nucleus IDA. Reason: Immuno-EM detected mouse Pifo in the nucleus as well as the Golgi/TGN. Testis fractionation put the long isoform (with an N-terminal coiled coil) in the nuclear fraction. No nuclear function is known, and the isoform-specific basis may not apply to all human isoforms. Retained as an observed location, non-core. Supporting Evidence: PMID:20643351 The ultrastructural analysis revealed that the protein is localized in the nucleus, Golgi apparatus, and in vesicles of the TGN PMID:20643351 we detected the long and short protein isoforms in the nuclear and cytoplasmic fraction, respectively, indicating that the N-terminal coiled-coil domain is important for nuclear localization |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasm, from UniProt subcellular location mapping. Reason: Pitchfork is a cytoplasmic vesicle/TGN and basal-body associated protein; cytoplasm is correct though general. Supporting Evidence: PMID:20643351 Pifo vesicles accumulate in the cytoplasm (Figure 5A, II) and localize to both the TGN and the growing axoneme |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | ACCEPT | Summary: Cytoplasm by ISS from mouse Pifo. Reason: Consistent with cytoplasmic vesicle localization shown for mouse and human PIFO. Supporting Evidence: PMID:20643351 Pifo vesicles accumulate in the cytoplasm (Figure 5A, II) and localize to both the TGN and the growing axoneme |
| GO:0005794 Golgi apparatus | IEA GO_REF:0000044 | ACCEPT | Summary: Golgi apparatus, UniProt subcellular location mapping. Reason: Pifo vesicles bud from the Golgi and Pifo localizes to the TGN. Supporting Evidence: PMID:20643351 In G0-deprived primary ciliated cells, Pifo localizes to the TGN and to the basal body |
| GO:0005802 trans-Golgi network | IEA GO_REF:0000107 | ACCEPT | Summary: trans-Golgi network, Ensembl Compara transfer from mouse. Reason: Directly observed for mouse Pifo in ciliated cells. Supporting Evidence: PMID:20643351 In G0-deprived primary ciliated cells, Pifo localizes to the TGN and to the basal body |
| GO:0005802 trans-Golgi network | ISS GO_REF:0000024 | ACCEPT | Summary: trans-Golgi network by ISS from mouse. Reason: Directly observed for mouse Pifo in ciliated cells. Supporting Evidence: PMID:20643351 In G0-deprived primary ciliated cells, Pifo localizes to the TGN and to the basal body |
| GO:0019894 kinesin binding | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Kinesin binding (KIF3A), Ensembl Compara transfer. Reason: KIF3A co-precipitates with PIFO in pull-downs. This supports association with the kinesin-2/IFT trafficking machinery, but direct binding and functional relevance are not established. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0019894 kinesin binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | KEEP AS NON CORE | Summary: Kinesin binding: KIF3A co-precipitated with tagged PIFO. Reason: Pull-down of endogenous KIF3A; consistent with vesicular ciliary transport, but not a core activity. Direct interaction is not shown. Supporting Evidence: PMID:20643351 We precipitated endogenous proteins using immobilized mouse and human SF-TAP-tagged PIFO in ciliated and cilia-disassembling HEK293T cells PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0019901 protein kinase binding | IEA GO_REF:0000107 | ACCEPT | Summary: Protein kinase binding (AURKA), Ensembl Compara transfer. Reason: AURKA association is central to the proposed mechanism of Pitchfork. Supporting Evidence: PMID:20643351 This pull-down analysis revealed that both WT and R80K PIFO physically interact with endogenous AurA |
| GO:0019901 protein kinase binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | ACCEPT | Summary: Protein kinase binding: PIFO pulls down endogenous Aurora A. Reason: Binding to AURKA is the mechanistic link to Pitchfork's role in activating Aurora A during cilia disassembly; both WT and R80K bind. Supporting Evidence: PMID:20643351 This pull-down analysis revealed that both WT and R80K PIFO physically interact with endogenous AurA PMID:20643351 these results indicate that mouse and human PIFO accumulate in a cell cycle-dependent manner at the basal body to activate AurA, a process that is specifically abolished by the R80K mutation |
| GO:0030295 protein kinase activator activity | IBA GO_REF:0000033 | ACCEPT | Summary: Protein kinase activator activity (IBA, PTN002051976). Reason: The PAINT node includes human CIMAP3 itself as experimental grounding, which is expected. Overexpressed PIFO prolongs AurA activation and the R80K mutant blocks it. Whether activation is direct (allosteric) or indirect is not resolved, but this is the best available MF for the AURKA-activating role. Supporting Evidence: PMID:20643351 overexpression of SF-TAP-PIFO during the early phase of cilia disassembly leads to a prolonged activation of AurA, whereas total AurA levels were largely unaffected PMID:20643351 overexpression of TAP-R80K PIFO completely blocked AurA phosphorylation |
| GO:0031267 small GTPase binding | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Small GTPase binding, Ensembl Compara transfer. Reason: RAB6, RAB8 and ARL13B co-precipitate with PIFO. This reflects association with ciliary targeting complexes, not a demonstrated core activity. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0031267 small GTPase binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | KEEP AS NON CORE | Summary: Small GTPase binding: RAB6A co-precipitated with PIFO. Reason: Pull-down association; direct binding and nucleotide dependence are untested. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0031267 small GTPase binding | IPI PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | KEEP AS NON CORE | Summary: Small GTPase binding: RAB8A co-precipitated with PIFO. Reason: Pull-down association; direct binding and nucleotide dependence are untested. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0031344 regulation of cell projection organization | IBA GO_REF:0000033 | ACCEPT | Summary: Regulation of cell projection organization (IBA). Reason: Correct but general. The specific process is primary cilium disassembly (added below as GO:0061523). Supporting Evidence: PMID:20643351 it is required specifically to control cilia retraction as well as the liberation and duplication of the basal body/centrosome during primary cilia disassembly |
| GO:0031344 regulation of cell projection organization | IEA GO_REF:0000107 | ACCEPT | Summary: Regulation of cell projection organization, Ensembl Compara transfer. Reason: Correct but general; see cilium disassembly. Supporting Evidence: PMID:20643351 it is required specifically to control cilia retraction as well as the liberation and duplication of the basal body/centrosome during primary cilia disassembly |
| GO:0031344 regulation of cell projection organization | ISS GO_REF:0000024 | ACCEPT | Summary: Regulation of cell projection organization by ISS from mouse. Reason: Correct but general; see cilium disassembly. Supporting Evidence: PMID:20643351 it is required specifically to control cilia retraction as well as the liberation and duplication of the basal body/centrosome during primary cilia disassembly |
| GO:0031410 cytoplasmic vesicle | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasmic vesicle, UniProt subcellular location mapping. Reason: PIFO decorates Golgi-derived vesicles that move along microtubules and the cilium. Supporting Evidence: PMID:20643351 Pifo vesicles accumulate in the cytoplasm (Figure 5A, II) and localize to both the TGN and the growing axoneme |
| GO:0031410 cytoplasmic vesicle | ISS GO_REF:0000024 | ACCEPT | Summary: Cytoplasmic vesicle by ISS from mouse. Reason: PIFO decorates Golgi-derived vesicles. Supporting Evidence: PMID:20643351 Pifo vesicles accumulate in the cytoplasm (Figure 5A, II) and localize to both the TGN and the growing axoneme |
| GO:0033674 positive regulation of kinase activity | IMP PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | ACCEPT | Summary: Positive regulation of kinase activity: PIFO enhances Aurora A activation. Reason: Overexpression of WT PIFO prolongs AurA activation, while R80K completely blocks AurA phosphorylation. This is the core regulatory output of Pitchfork. Supporting Evidence: PMID:20643351 overexpression of SF-TAP-PIFO during the early phase of cilia disassembly leads to a prolonged activation of AurA, whereas total AurA levels were largely unaffected PMID:20643351 overexpression of TAP-R80K PIFO completely blocked AurA phosphorylation PMID:20643351 these results indicate that mouse and human PIFO accumulate in a cell cycle-dependent manner at the basal body to activate AurA, a process that is specifically abolished by the R80K mutation |
| GO:0036064 ciliary basal body | IBA GO_REF:0000033 | ACCEPT | Summary: Ciliary basal body (IBA). Reason: PIFO accumulates at the basal body, especially during cilia disassembly. Supporting Evidence: PMID:20643351 Restimulation of G0 cells led to rapid accumulation of PIFO-Venus at the basal body/centrosome PMID:20643351 In G0-deprived primary ciliated cells, Pifo localizes to the TGN and to the basal body |
| GO:0036064 ciliary basal body | IEA GO_REF:0000107 | ACCEPT | Summary: Ciliary basal body, Ensembl Compara transfer. Reason: Supported by mouse and human data. Supporting Evidence: PMID:20643351 Restimulation of G0 cells led to rapid accumulation of PIFO-Venus at the basal body/centrosome |
| GO:0036064 ciliary basal body | ISS GO_REF:0000024 | ACCEPT | Summary: Ciliary basal body by ISS from mouse. Reason: Supported by mouse and human data. Supporting Evidence: PMID:20643351 Restimulation of G0 cells led to rapid accumulation of PIFO-Venus at the basal body/centrosome |
| GO:0043015 gamma-tubulin binding | IDA PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | KEEP AS NON CORE | Summary: Gamma-tubulin binding from pull-down. Reason: Gamma-tubulin co-precipitates with PIFO. This is consistent with basal body/PCM proximity, but a lysate pull-down does not show direct binding and it is not a core function. Supporting Evidence: PMID:20643351 We precipitated endogenous proteins using immobilized mouse and human SF-TAP-tagged PIFO in ciliated and cilia-disassembling HEK293T cells PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0043015 gamma-tubulin binding | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Gamma-tubulin binding, Ensembl Compara transfer. Reason: See IDA row; association only. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0044782 cilium organization | IBA GO_REF:0000033 | ACCEPT | Summary: Cilium organization (IBA). Reason: Correct; Pitchfork's specific role is in cilium disassembly rather than assembly. Supporting Evidence: PMID:20643351 it is required specifically to control cilia retraction as well as the liberation and duplication of the basal body/centrosome during primary cilia disassembly PMID:20643351 haploinsufficiency for Pifo does not block cilia assembly per se |
| GO:0048487 beta-tubulin binding | IDA PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | KEEP AS NON CORE | Summary: Beta-tubulin binding from pull-down. Reason: Beta-tubulin co-precipitates with PIFO, consistent with microtubule/vesicle association; direct binding is not shown. Supporting Evidence: PMID:20643351 We precipitated endogenous proteins using immobilized mouse and human SF-TAP-tagged PIFO in ciliated and cilia-disassembling HEK293T cells PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0048487 beta-tubulin binding | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Beta-tubulin binding, Ensembl Compara transfer. Reason: See IDA row; association only. Supporting Evidence: PMID:20643351 the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein Ξ³-Tubulin, the centriolar protein Centrin, the MT protein Ξ²-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture |
| GO:0061523 cilium disassembly | IMP PMID:20643351 Pitchfork regulates primary cilia disassembly and left-right... | NEW | Summary: Pitchfork is required for primary cilium retraction and basal body release during cilia disassembly. Reason: Pifo haploinsufficient cells and cells expressing dominant R80K PIFO fail to retract the cilium and release the basal body when quiescent cells re-enter the cell cycle; cilium assembly is not blocked. Participation: PIFO accumulates at the basal body and drives AURKA activation, the kinase step that triggers HDAC6-mediated axoneme destabilization. Comparator check: NEDD9, the other AURKA activator in this pathway, together with AURKA and HDAC6, carries GO:0061523 involved_in in human GOA (PMID:17604723). The term is therefore used for activators of the disassembly kinase. Evidence is mouse plus human-construct data from a single study. Supporting Evidence: PMID:20643351 the basal body fails to disconnect from the cilium and the cilium is retained during mitosis PMID:20643351 it is required specifically to control cilia retraction as well as the liberation and duplication of the basal body/centrosome during primary cilia disassembly PMID:20643351 These results indicate that PIFO regulates AurA and is required for several aspects of cilia disassembly PMID:20643351 haploinsufficiency for Pifo does not block cilia assembly per se |
| GO:0061512 protein localization to cilium | ISS PMID:26901434 Pitchfork and Gprasp2 Target Smoothened to the Primary Ciliu... | NEW | Summary: Pifo is an essential component of a Hedgehog-induced ciliary targeting complex that moves Smoothened into the primary cilium. Reason: In mouse limb-bud cells, conditional Pifo deletion after cilia had formed nearly abolished SAG-induced Smo entry into cilia and Gli1/Gli2 induction; Venus-Pifo rescued both. Participation: Pifo is a structural member of the Pifo-Gprasp2-Smo targeting complex, with GPRASP2 bridging to the Smo ciliary targeting motif. It contributes the complex that carries out the step, rather than being cargo. Human PIFO forms the complex when GPRASP2 is co-expressed. Evidence is from mouse knockout cells, hence ISS. Supporting Evidence: PMID:26901434 Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC PMID:26901434 Whereas 70β80% of Pifowt/wt PLCs show efficient Smo translocation to the PC within 30 min of ligand exposure, PifoFD/FD PLCs with normally formed PC barely responded to SAG induced pathway activation, which can be sufficiently rescued by stable Venus-Pifo expression PMID:26901434 suggesting that a heterotrimeric ciliary targeting complex can be formed in which Gprasp2 bridges between Smo and Pifo file:human/CIMAP3/CIMAP3-deep-research-falcon.md Direct binding was demonstrated for GPRASP2βSMO, not PIFOβSMO; most causal evidence is from mouse cells and tagged proteins. |
| GO:0097545 axonemal doublet microtubule | IDA PMID:39743588 Structural diversity of axonemes across mammalian motile cil... | NEW | Summary: Cryo-EM places CIMAP3 at the external surface of axonemal doublet microtubules in all mammalian motile axonemes examined. Reason: Comparative cryo-EM of mammalian motile cilia (sperm, oviduct, respiratory, ependymal) assigns CIMAP3 density to a protofilament cleft on the doublet exterior. This explains the 'ciliary microtubule-associated protein' name and the SHIPPO-repeat relationship to ODF3/CIMAP1. It is a location annotation; no mechanical function is established. Supporting Evidence: PMID:39743588 For example, CIMAP3 is present in all mammalian axonemes hitherto studied, yet CIMAP2, which binds the same protofilament cleft, is found only in sperm PMID:39743588 ciliary microtubule-associated proteins (CIMAPs) bound close to the external surface of the DMT |
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Download this section (compressed HTML)Q: Does PIFO activate Aurora A directly (e.g. allosterically, like TPX2 or NEDD9), or indirectly by delivering an activator or scaffold to the basal body?
Q: Which, if any, of the pull-down partners (RAB6, RAB8, ARL13B, KIF3A, centrin, tubulins) bind PIFO directly, and are they needed for its basal-body accumulation?
Q: Is CIMAP3 relevant to cilium disassembly in adult human ciliated cells, or mainly in embryonic organizer cells and sperm (consistent with its absence from the HPA cilium atlas)?
Q: Is the R80K variant causal for laterality/ciliopathy phenotypes in humans?
Q: Is the axonemal-doublet CIMAP3 pool in motile cilia (Leung et al. 2025) functionally related to the transient basal-body/vesicular pool that regulates disassembly and Smo trafficking in primary cilia?
Q: Does human CIMAP3 participate in SMO ciliary entry, as shown for mouse Pifo with GPRASP2?
Experiment: Reconstitute AURKA kinase activity with purified PIFO (WT vs R80K) to test direct activation.
Experiment: CRISPR knockout of CIMAP3 in a ciliated human line expressing it, followed by serum-induced cilia disassembly assays and measurement of AURKA T288 phosphorylation at the basal body.
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