Background: Connecdenns are GEFs for Rab35, a GTPase controlling endocytic recycling. Regulation of connecdenn function is unknown. Results: The GEF activity of connecdenn 1 and 2 are autoinhibited, and this is regulated by Akt activation downstream of insulin stimulation. Conclusion: Phosphorylation of connecdenn by Akt may provide a mechanism to control cargo recycling. Significance: Signaling pathways impinge on membrane-trafficking pathways.

Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

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.

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Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brain. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn and allosterically enhances its GEF activity towards Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals upregulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking in brain tumors.

Epileptic encephalopathies comprise a clinically and genetically heterogenous group of disorders characterized by global developmental delay and ongoing seizure activity. It is generally considered that seizure activity is the primary pathology and leads to altered cognitive function. However, epileptic encephalopathy can also result from primary defects in neurodevelopment that lead to seizures. Here we examine the symptomology of individuals with epileptic encephalopathy resulting from biallelic pathogenic variants in DENND5A, encoding a protein involved in intracellular membrane trafficking. We established a cohort of 15 individuals from 14 families with 21 unique variants in DENND5A and analyzed phenotypic surveys answered by their treating clinicians. We obtained cells derived from several cohort members and examined DENND5A expression and stability, and we examined the role of DENND5A in symmetric cell division capability. Finally, we generated a mouse line expressing a homozygous mutation found in the cohort and analyzed brain morphology in vivo using a 7 Tesla magnetic resonance imaging system. Our cohort study reveals that global developmental delay, gross abnormalities in brain development including ventriculomegaly and corpus callosum dysgenesis, seizures, microcephaly and hypotonia are found in the majority of the cohort. Patient-derived neural precursor cells from induced pluripotent stem cells, as well as patient-derived lymphoblasts, display loss of DENND5A protein and exhibit problems with symmetric cell division, an essential process in early neural development. The introduction of a homozygous human point mutation into the conserved sequence in mouse leads to an animal with reduced brain volume and widespread regional brain dysgenesis and ventriculomegaly, phenotypes shared with the human cohort. Taken together, our multi-dimensional study establishes DENND5A as a critical gene during neurodevelopment and that biallelic loss of function variants, including missense and truncating variants as well as intronic splice site variants, result in a developmental epileptic encephalopathy.

Cytokinesis is the final stage of cell division. Successful cytokinesis requires membrane trafficking pathways regulated by Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). Late in cytokinesis, an intercellular cytokinetic bridge (ICB) connecting the two daughter cells undergoes abscission, which requires depolymerization of actin. Rab35 recruits MICAL1 to oxidate and depolymerize actin filaments. We report that DENND2B, a protein previously implicated in cancer, mental retardation and multiple congenital disorders functions as a GEF for Rab35 and recruits and activates the GTPase at the ICB. Unexpectedly, the N terminal region of DENND2B interacts with an active mutant of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission resulting in increased multinucleated cells and over-accumulation of F-actin at the ICB. F-actin accumulation leads to formation of a chromatin bridge, a process known to activate the NoCut/abscission checkpoint, and DENND2B knockdown actives Aurora B kinase, a hallmark of checkpoint activation. This study identifies DENND2B as a crucial player in cytokinetic abscission and provides insight into the multisystem disorder associated with DENND2B mutation.