Gene Ontology annotation through association of InterPro records with GO terms
Annotation inferences using phylogenetic trees
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
Gene Ontology annotation based on curation of immunofluorescence data
Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara
Combined Automated Annotation using Multiple IEA Methods
D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.
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Dual specificity was established here, with the active fragment binding RI-alpha, RII-alpha and RII-beta but not RI-beta, and the binding site on the R subunits mapped to their N-terminal dimerization domain.
"Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha."
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The RGS assignment was homology-only from the outset, covering residues 70-215 of the 372-residue mouse clone.
"Surprisingly, however, residues 70–215 showed homology to the RGS domain found in RGS proteins."
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The authors themselves recorded that the RGS domain lacks conserved charged residues and predicted it would not behave like a canonical RGS protein, the earliest warning against reading GAP activity off the domain name.
"Surprisingly, not all of the charged residues conserved in RGS proteins reported so far were conserved in D-AKAP2."
Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein.
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The full-length human protein was cloned here and shown to be 662 residues with a second RGS domain not present in the original mouse clone.
"Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues)."
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Endogenous protein from three species including human was reported as highly enriched in mitochondria. This is the source of the gene's UniProt name and of the mitochondrion annotations, and it is the claim that PMID:19797056 later disputed.
"Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria."
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In vivo association with PKA was shown by cAMP-agarose pull-down from mouse brain. This is the experiment behind the mouse GO:0043495 IDA that both the PAINT and Compara rows transfer.
"In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay."
Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS).
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Hydrogen-deuterium exchange showed the protein is two folded regions separated by disordered linkers, with the PKA-binding site highly protected and the PDZ-binding motif solvent-exposed, the physical basis for the two C-terminal motifs behaving independently.
"The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2."
PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells.
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The C-terminal PDZ motif binds PDZK1 tightly and NHERF-1 about fourfold more weakly, linking AKAP10 to the scaffolds that position the NaPi-IIa phosphate cotransporter.
"In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity."
Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation.
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The common human 646V variant is associated with increased basal heart rate and decreased heart rate variability. This is a population association, not a molecular function or a process of the wild-type protein, and no cardiac GO term is proposed from it.
"We also found that a common variant of AKAP10 in humans (646V, 40% of alleles) was associated with increased basal heart rate and decreased heart rate variability (markers of low cholinergic/vagus nerve sensitivity)."
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The mouse allele is a C-terminal gene trap that the authors read as dominant-interfering, so it is not a clean loss of function for the wild-type protein.
"In both heterozygous and homozygous mutant mice derived from these mESCs, the same Akap10 disruption increases the cardiac response to cholinergic signals, suggesting a dominant interfering effect of the Akap10 mutant allele."
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The authors state that the molecular mechanism is unknown.
"Although the molecular mechanism remains unknown, our findings in mutant mESCs, mice, and a common human AKAP10 SNP all suggest a role for AKAP10 in heart rhythm control."
D-AKAP2 interacts with Rab4 and Rab11 through its RGS domains and regulates transferrin receptor recycling.
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The tandem RGS domains bind Rab11 and GTP-bound Rab4, the first report of RGS domains binding small GTPases.
"We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases."
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The two RGS domains act as one binding unit, since residues 120-513 are necessary and sufficient while neither domain alone binds either Rab.
"Neither RGS-A (residues 120–369) nor RGS-B (residues 374–513) alone was able to interact with Rab4 or Rab11, suggesting that the two RGS domains form a single functional unit."
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The paper states the negative result for the canonical RGS function directly, with no specific interaction with heterotrimeric G proteins and no ability to activate them.
"D-AKAP2 has so far demonstrated no specific interactions with heterotrimeric G proteins, and no ability to activate them, raising the possibility of nontraditional targets."
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Endogenous human AKAP10 is almost entirely cytosolic in HeLa cells, contradicting the earlier mitochondrial enrichment report.
"Cell fractionation showed that endogenous D-AKAP2 is almost entirely cytosolic"
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Knockdown accelerates transferrin recycling by 73 percent, establishing that the wild-type protein restrains traffic out of the recycling compartment.
"Fitting all of the data to an exponential decay function yielded a rate of transferrin recycling that was 73 ± 21% faster in the D-AKAP2 knockdown cells than in control cells"
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All constructs used the human gene, which is what makes human experimental evidence codes appropriate for the rows proposed from this paper.
"All D-AKAP2 constructs used the human gene with valine at position 646."
Structure of D-AKAP2:PKA RI complex: insights into AKAP specificity and selectivity.
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Crystal structure of the AKAP10 anchoring helix bound to the PKA RI-alpha dimerization/docking domain, the direct structural demonstration that this protein anchors type I PKA.
"Here we have determined the structures of the RIalpha D/D domain alone and in complex with D-AKAP2."
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Dual specificity is explained by a shift in helical register between the RI and RII complexes, so the same helix binds both R subunit types in different ways.
"An unexpected shift in the helical register of D-AKAP2 compared to the RIIalpha:D-AKAP2 complex structure makes the mode of binding to RIalpha novel."
A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs.
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AKAP anchoring works through a short helical region binding the R-subunit dimerization domain, and this study measured those affinities biophysically.
"The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far."
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The binding measurements are fluorescence anisotropy, supported by HADDOCK modelling and cellular colocalisation, so the IPI rows rest on a direct affinity assay.
"To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies."
D-AKAP2:PKA RII:PDZK1 ternary complex structure: insights from the nucleation of a polyvalent scaffold.
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A single C-terminal segment, disordered on its own, presents an alpha-helix to PKA and a beta-strand to PDZK1, one stretch of chain performing two different structural roles.
"Here, we describe a structure of D-AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α-helix to PKA and a β-strand to PDZK1."
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The two arms are ordered, because forming the PKA complex is a prerequisite for high-affinity PDZK1 binding, so the scaffold assembles in sequence rather than independently.
"Formation of the D-AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon."
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Unlike most AKAPs, AKAP10 does not contact the membrane protein itself; it reaches transporters through the PDZ scaffold. This is why its membrane annotation should be read as indirect.
"In contrast to many other AKAPs, D-AKAP2 does not interact directly with the membrane protein."
Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.
Quantitative fragmentomics allow affinity mapping of interactomes.
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A proteome-scale affinity survey of PDZ domains against PDZ-binding motifs. The 21 AKAP10 rows it contributes are cells of this matrix, measurements of the AKAP10 motif binding a PDZ domain, not demonstrations of 21 cellular complexes.
"Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer."
Multimodal cell maps as a foundation for structural and functional genomics.
Dual-specific AKAPs bind type I and II PKA regulatory subunits
Affinage mechanistic annotation for AKAP10 (human)
AKAP10 RGS domains: are the Galpha GAP contact residues present?
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Both AKAP10 RGS domains retain 0 of 5 RGS4 Galpha transition-state contact residues, scoring like the AXIN1 negative control and unlike the RGS16 and RGS9 positive controls, which retain 5 of 5 each.
"| AKAP10 RGS-A | subject | 30.3% | 0/5 | P315 | no |"
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The decisive asparagine, RGS4 Asn128, whose N128A substitution abolishes GAP activity, is replaced by proline in RGS-A and by glycine in RGS-B.
"| AKAP10 RGS-B | subject | 24.3% | 0/5 | G452 | no |"