ABSTRACT
The inositol polyphosphate 5'-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.
Subject(s)
Cilia/metabolism , Phosphoric Monoester Hydrolases/metabolism , Animals , Cells, Cultured , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Dyneins/metabolism , Mice , Microscopy, Fluorescence , Phosphoric Monoester Hydrolases/geneticsABSTRACT
The prenyl binding protein PDEδ enhances the diffusion of farnesylated Ras proteins in the cytosol, ultimately affecting their correct localization and signaling. This has turned PDEδ into a promising target to prevent oncogenic KRas signaling. In this review we summarize and describe the structure-guided-development of the three different PDEδ inhibitor chemotypes that have been documented so far. We also compare both their potency for binding to the PDEδ pocket and their in vivo efficiency in suppressing oncogenic KRas signaling, as a result of the inhibition of the PDEδ/KRas interaction.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Animals , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Enzyme Inhibitors/chemistry , Pyridazines/chemistry , Pyridazines/pharmacologyABSTRACT
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.
Subject(s)
Cilia/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Phosphoric Monoester Hydrolases/metabolism , ADP-Ribosylation Factors/chemistry , ADP-Ribosylation Factors/metabolism , Animals , Cell Line , Fluorescence Polarization , Green Fluorescent Proteins/metabolism , Guanosine Triphosphate/metabolism , Inositol Polyphosphate 5-Phosphatases , Kinetics , Mice , Models, Biological , Monomeric GTP-Binding Proteins/metabolism , Mutant Proteins/metabolism , Neuropeptides/metabolism , Protein Binding , Protein Prenylation , Protein Sorting Signals , Protein Structure, Secondary , Protein Transport , Ras Homolog Enriched in Brain ProteinABSTRACT
The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414-530) and CC2 (aa 530-630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.
Subject(s)
Aurora Kinase A/metabolism , Carrier Proteins/metabolism , Centrosome/metabolism , Fetal Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis/physiology , Animals , Aurora Kinase A/genetics , Carrier Proteins/genetics , Fetal Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Mice , Microtubule-Associated Proteins/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Binding/physiology , Protein Structure, TertiaryABSTRACT
During the mitotic division cycle, cells pass through an extensive microtubule rearrangement process where microtubules forming the mitotic spindle apparatus are dynamically instable. Several centrosomal- and microtubule-associated proteins are involved in the regulation of microtubule dynamics and stability during mitosis. Here, we focus on members of the transforming acidic coiled coil (TACC) family of centrosomal adaptor proteins, in particular TACC3, in which their subcellular localization at the mitotic spindle apparatus is controlled by Aurora-A kinase-mediated phosphorylation. At the effector level, several TACC-binding partners have been identified and characterized in greater detail, in particular, the microtubule polymerase XMAP215/ch-TOG/CKAP5 and clathrin heavy chain (CHC). We summarize the recent progress in the molecular understanding of these TACC3 protein complexes, which are crucial for proper mitotic spindle assembly and dynamics to prevent faulty cell division and aneuploidy. In this regard, the (patho)biological role of TACC3 in development and cancer will be discussed.
Subject(s)
Adaptor Protein Complex 3/chemistry , Centrosome/chemistry , Microtubule-Associated Proteins/chemistry , Mitosis , Adaptor Protein Complex 3/genetics , Adaptor Protein Complex 3/physiology , Animals , Cell Division/genetics , Centrosome/pathology , Centrosome/physiology , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Mitosis/genetics , Multigene Family/genetics , Protein Structure, Tertiary/genetics , Spindle Apparatus/geneticsABSTRACT
Plexin-B1 regulates various cellular processes interacting directly with several Rho proteins. Molecular details of these interactions are, however, not well understood. In this study, we examined in vitro and in silico the interaction of the Rho binding domain (B1RBD) of human Plexin-B1 with 11 different Rho proteins. We show that B1RBD binds in a GTP-dependent manner to Rac1, Rac2, Rac3, Rnd1, Rnd2, Rnd3, and RhoD, but not to RhoA, Cdc42, RhoG, or Rif. Interestingly, Rnd1 competitively displaces the Rac1 from B1RBD but not vice versa. Structure-function analysis revealed a negatively charged loop region, called B1L(31), which may facilitate a selective B1RBD interaction with Rho proteins.
Subject(s)
Nerve Tissue Proteins/chemistry , Receptors, Cell Surface/chemistry , rho GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Binding, Competitive , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Sequence Homology, Amino Acid , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
Major advances have been made in understanding the structure, function and regulation of the small GTP-binding proteins of the Rho family and their involvement in multiple cellular process and disorders. However, intrinsic nucleotide exchange and hydrolysis reactions, which are known to be fundamental to Rho family proteins, have been partially investigated in the case of RhoA, Rac1 and Cdc42, but for others not at all. Here we present a comprehensive and quantitative analysis of the molecular switch functions of 15 members of the Rho family that enabled us to propose an active GTP-bound state for the rather uncharacterized isoforms RhoD and Rif under equilibrium and quiescent conditions.
