Christopher J. Westlake

A Core Complex of BBS Proteins Cooperates with the GTPase Rab8 to Promote Ciliary Membrane Biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH2-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11–Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogene

The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G1 cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11–Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.

Primary cilia signaling mediates intraocular pressure sensation.

Significance This study defines a cellular mechanism by which primary cilia mediate mechanosensation in intraocular pressure regulation. Changes in pressure are sensed by the interaction of the inositol phosphatase OCRL with transient receptor potential vanilloid 4 (TRPV4), a primary cilia-based calcium channel. Pediatric glaucoma (Lowe) syndrome patient cells with defective OCRL failed to respond to agonists of TRPV4, and targeting of TRPV4 lowered intraocular pressure in vivo. These findings significantly advance the current understanding of how intraocular pressure is regulated. Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-β, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.

Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

Identification of methionine aminopeptidase-2 (MetAP-2) as the molecular target of the antiangiogenic compound TNP-470 has sparked interest in N-terminal Met excisions (NME) role in endothelial cell biology. In this regard, we recently demonstrated that MetAP-2 inhibition suppresses Wnt planar cell polarity (PCP) signaling and that endothelial cells depend on this pathway for normal function. Despite this advance, the substrate(s) whose activity is altered upon MetAP-2 inhibition, resulting in loss of Wnt PCP signaling, is not known. Here we identify the small G protein Rab37 as a MetAP-2-specific substrate that accumulates in the presence of TNP-470. A functional role for aberrant Rab37 accumulation in TNP-470s mode of action is demonstrated using a Rab37 point mutant that is resistant to NME, because expression of this mutant phenocopies the effects of MetAP-2 inhibition on Wnt PCP signaling-dependent processes.

Abstract A35: Oncogenic Ras signaling involves formation of perinuclear CK2α nd p-ERK1/2 signaling complexes that require the KSR-1 scaffold protein and endosomal trafficking

Background: Ras GTPases play critical roles in transmitting signals from extracellular growth factors (GFs) to downstream effector pathways. Oncogenic Ras mutations occur frequently in human tumors and encode active Ras proteins that promote deregulated signaling in a GF-independent manner. Although the differences between oncogenic Ras and GF-driven signal transmission have been studied extensively, further insights into the features that define normal and tumorigenic Ras signaling may reveal important targets for development of novel anti-cancer therapies. Expression of oncogenic Ras in primary cells can provoke oncogene-induced senescence (OIS), an intrinsic tumor suppressor mechanism that is bypassed in cancer cells. The transcription factor C/EBPβ is post-translationally activated by Ras signaling, and activated C/EBPβ contributes to OIS in MEFs. We previously reported that C/EBPβ activation by H-RasV12 signaling is suppressed in transformed cells, but not in primary cells, by its 39UTR. This novel RNA-mediated control mechanism is termed 3’UTR regulation of protein activity (UPA). A sequence in the 3’UTR regulates localization of Cebpb transcripts, directing them to a peripheral cytoplasmic region that is distinct from a perinuclear region containing the C/EBPβ activating kinase, p-ERK. Thus, UPA blocks C/EBPβ activation in tumor cells and suppresses its pro-senescence functions. These findings suggest an important role for subcellular localization of downstream kinases in Ras signal transmission. In the present study we have investigated the mechanism and functional consequences of perinuclear compartmentalization of the Ras effector kinases p-ERK and CK2α. Results: We identified CK2α as a Ras effector kinase that phosphorylates C/EBPβ on a Ser residue in its DNA-binding domain. This modification can be blocked by the Cebpb 39UTR in transformed/immortalized cells. Like p-ERK, CK2α localizes to the perinuclear region in cells expressing oncogenic Ras or BRAFV600E. The MAPK signaling scaffold KSR-1 is essential for perinuclear targeting of p-ERK and CK2α, which can also be blocked by inhibitors of MEK or CK2. KSR-1 itself is also sequestered in the perinuclear region in response to oncogenic Ras-Raf signals. In addition, endocytic trafficking is required for perinuclear targeting of KSR-1, p-ERK and CK2α. CK2α co-localized with KSR-1 and Rab11 (a marker of recycling endosomes), while p-ERK was associated with a different KSR-1 positive endosomal population. Interestingly, we found that whereas oncogenic Ras induced constitutive perinuclear localization of p-ERK and CK2α in both transformed and senescing cells, GFs transiently elicited perinuclear compartmentalization of these kinases with delayed kinetics (4-6 hrs post-stimulation). Conclusions: We propose that oncogenic Ras signaling constitutively activates the delayed phase of GF signaling in which the effector kinases p-ERK and CK2α form KSR-1-dependent perinuclear signaling complexes (PSCs). In this subcellular location, ERK and CK2 and perhaps other kinases can access critical targets that promote oncogenic transformation or senescence. These distinct cellular responses may be controlled in part by differential localization of mRNAs encoding substrates such as C/EBPβ. PSCs were observed across a range of human cancer cell lines and in mouse lung tumor tissue, supporting a critical role for localized signaling in driving tumorigenesis. Citation Format: Sandip K. Basu, Sook Lee, Krisada Sakchaisri, Vijay Walia, Christopher J. Westlake, Deborah K. Morrison, Peter F. Johnson. Oncogenic Ras signaling involves formation of perinuclear CK2α nd p-ERK1/2 signaling complexes that require the KSR-1 scaffold protein and endosomal trafficking. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A35.