A cell-based GEF assay reveals new substrates for DENN domains and a role for DENND2B in primary ciliogenesis

Abstract

Primary cilia are sensory antennae crucial for cell and organism development, and defects in their biogenesis cause ciliopathies. Ciliogenesis involves membrane trafficking mediated by small guanosine triphosphatases (GTPases) including Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). The largest family of Rab GEFs is the DENN domain–bearing proteins. Here, we screen all 60 Rabs against two major DENN domain families using a cellular GEF assay, uncovering 19 novel DENN/Rab pairs. The screen reveals Rab10 as a substrate for DENND2B, a protein previously implicated in cancer and severe mental retardation. Through activation of Rab10, DENND2B represses the formation of primary cilia. Through a second pathway, DENND2B functions as a GEF for RhoA to control the length of primary cilia. This work thus identifies an unexpected diversity in DENN domain–mediated activation of Rabs, a previously unidentified non-Rab substrate for a DENN domain, and a new regulatory protein in primary ciliogenesis.

1-L Transcription in Alzheimer’s Disease

“…DENND2C (DENN domain containing 2C) is guanine nucleotide exchange factor for RAB8A, RAB8B, RAB10, RAB15, and RAB35 [ 78 ]. The DENN domain-bearing proteins comprise the largest family of Rab GEFs [ 78 ].…”

“…DENND2C (DENN domain containing 2C) is guanine nucleotide exchange factor for RAB8A, RAB8B, RAB10, RAB15, and RAB35 [ 78 ]. The DENN domain-bearing proteins comprise the largest family of Rab GEFs [ 78 ]. In a study of microglial depletion and repopulation with new and unprimed microglia in aged mice, normally age-decreased gene Dennd2c was reversed after microglial repopulation [ 79 ].…”

The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization

“…Since it is the membrane-bound active form of Rab10 which is phosphorylated by LRRK2 ( Liu et al, 2018 ; Gomez et al, 2019 ; Lara Ordóñez et al, 2019 ; Fdez et al, 2022 ), it will be interesting to determine whether membrane-bound vesicular structures containing phospho-Rab10 accumulate at the subdistal appendage of the mother centriole in pathogenic LRRK2-expressing cells. In addition, and given recent reports of the localization of active Rab10 at the mother centriole in a manner regulated by the GDP/GTP exchange factor DENND2B ( Kumar et al, 2022 ), future studies should address whether the pathogenic LRRK2-mediated centriolar phospho-Rab10 accumulation and the resulting cohesion deficits are modulated by DENND2B.…”

DENND2B activates Rab35 at the intercellular bridge regulating cytokinetic abscission and tetraploidy

“…For example, when GEFs are artificially targeted to organelles such as mitochondria, Rab substrates relocalize to these new sites [44]. Utilizing this approach, we discovered that DENN domain proteins activate a larger array of Rab substrates than previously thought [45]. Through investigation of newly identified DENN/Rab GEF/substrate pairs we discovered that DENND2B activates Rab10 to regulate primary cilia formation [45].…”

“…Utilizing this approach, we discovered that DENN domain proteins activate a larger array of Rab substrates than previously thought [45]. Through investigation of newly identified DENN/Rab GEF/substrate pairs we discovered that DENND2B activates Rab10 to regulate primary cilia formation [45]. DENND2B is multifunctional in that it also acts as a GEF for Rab13, promoting the development of epithelial cancer [42].…”