Synthesis of the Rheb and K‐Ras4B GTPases

Chen, Yong-Xiang; Koch, Sebastian; Uhlenbrock, Katharina; Weise, Katrin; Das, Debapratim; Gremer, Lothar; Brunsveld, Luc; Wittinghofer, Alfred; Winter, Roland; Triola, Gemma; Waldmann, Herbert

doi:10.1002/anie.201001884

Cited by 73 publications

(93 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In contrast, the affinity between PDE6δ and the farnesylated C-terminal peptide of Rheb falls into the submicromolar range ( K d =445±83 nM,±indicates s.d., n =10) (Fig. 1b; right), which is in the same range with the previously described values1213. These data raised the question, whether the almost 100-fold higher affinity of INPP5E towards PDE6δ as compared to Rheb is involved in the sorting mechanism of these two proteins to different destinations.…”

Section: Resultssupporting

confidence: 85%

“…Given that the prenyl-binding protein PDE6δ is the shuttle factor mediating the localization of INPP5E and Rheb1316182526, we set out to characterize the interaction of PDE6δ with INPP5E and Rheb. Previously we have shown that farnesylated C-terminal peptides derived from Rheb or KRas bind to PDE6δ in exactly the same way and with similar affinities as the full-length farnesylated proteins1213. Hence, we used a fluorescently labelled C-terminal farnesylated and carboxy-methylated peptide of INPP5E (residues 637–644) and Rheb (residues 175–181) to measure the affinity to PDE6δ by fluorescence polarization.…”

Section: Resultsmentioning

confidence: 99%

“…It was found as a solubilizing factor for the prenylated subunits of this enzyme and was later shown to be a general prenyl-binding protein (hence also called PrBP/PDE6δ)891011. PDE6δ was shown to bind prenylated peptides or proteins of the Ras subfamily with approximately micromolar affinity1213 and to play a critical role in their cellular distribution141516. Since it is believed to be crucial for the localization and thus the activity of the oncoprotein Ras, inhibitors of the Ras-PDE6δ complex were actually considered as promising Ras drug candidates17.…”

mentioning

confidence: 99%

See 2 more Smart Citations

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

et al. 2016

Self Cite

View full text Add to dashboard Cite

The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination.

show abstract

Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…These results show that the presence of both the methyl and farnesyl modifications increases the affinity between KRAS4b and PDEδ close to two orders of magnitude compared with only KRAS4b-Far. Previous studies using a fluorescence-polarization (FP) assay showed a 10-fold higher binding affinity (K d = 0.3 μM) between KRAS4b and PDEδ (29). This discrepancy is likely caused by the use of different techniques (analytical ultracentrifugation and ITC vs. FP), different assay conditions, and the use of semisynthetic and refolded KRAS4b vs. recombinant fully processed KRAS4b that we used for this study.…”

Section: The Role Of Farnesyl and Methyl Groups In Interactions Betweenmentioning

confidence: 82%

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

Dharmaiah

Bindu

Tran

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

157

197

View full text Add to dashboard Cite

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

show abstract

“…GSF=GDI solubilising factor, NA=not available and text in red indicates that the prediction has not been verified experimentally yet. References are [8,9,22,38,43,44].…”

Section: Open Questions and Future Perspectivesmentioning

confidence: 99%

Shuttling and sorting lipid-modified cargo into the cilia

Stephen

Ismail

2016

Biochemical Society Transactions

View full text Add to dashboard Cite

Primary cilia are hair-like microtubule-based organelles that can be found on almost all human cell types. Although the cilium is not separated from the cell by membranes, their content is different from that of the cell body and their membrane composition is distinct from that of the plasma membrane. Here we will introduce a molecular machinery that shuttles and sorts lipid modified proteins to the cilium, thus contributing in maintaining its distinct composition. The mechanism involves the binding of the GDI-like solubilising factors, UNC119a, UNC119b and PDE6D, to the lipid modified ciliary cargo and the specific release of the cargo in the cilia by the ciliary small G protein Arl3 in a GTP dependent manner.

show abstract

Synthesis of the Rheb and K‐Ras4B GTPases

Cited by 73 publications

References 44 publications

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

Shuttling and sorting lipid-modified cargo into the cilia

Contact Info

Product

Resources

About