Abstract
The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking. Proteins bearing a differentially expressed in normal and neoplastic cells (DENN) domain have emerged as the largest family of Rab-activating guanine nucleotide exchange factors (GEFs). Rab12 functions in the initiation of starvation-induced autophagy, and our previous work revealed that its activator, DENN domain-containing protein 3 (DENND3), is phosphorylated and activated upon starvation. However, how the GEF activity of DENND3 toward Rab12 is regulated at the molecular level is still not understood. Here, we combine size-exclusion chromatography, Förster resonance energy transfer, pulldown, and in vitro GEF assays to demonstrate that regulation of GEF activity is achieved through an intramolecular interaction that is controlled by a key residue in DENND3, tyrosine 940. Our study sheds light on the regulation of Rab12 activation and lays the groundwork for characterizing the regulation of other DENN domain-containing proteins.
Highlights
The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking
They are involved in diverse biological functions [4], and mutations in several differentially expressed in normal and neoplastic cells (DENN) domain-bearing proteins are linked to human diseases, including the tumor suppressor folliculin associated with Birt-HoggDubé syndrome [9], C9orf72 linked to familial frontotemporal dementia and amyotrophic lateral sclerosis [10, 11], and DENND1B associated with childhood asthma [12]
We demonstrate that phosphorylation of a specific tyrosine residue in DENN domain-containing protein 3 (DENND3) regulates this intramolecular interaction, suggesting that a signaling pathway involving a tyrosine kinase impinges upon the intramolecular interaction to regulate DENND3 guanine nucleotide exchange factors (GEFs) activity toward Rab12
Summary
The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking. Proteins bearing a differentially expressed in normal and neoplastic cells (DENN) domain have emerged as the largest family of Rab-activating guanine nucleotide exchange factors (GEFs). Rab functions in the initiation of starvation-induced autophagy, and our previous work revealed that its activator, DENN domain-containing protein 3 (DENND3), is phosphorylated and activated upon starvation. We recently demonstrated that DENN domain-containing protein 3 (DENND3) functions in starvation-induced macroautophagy [13, 14]. ULK phosphorylates DENND3 at Ser554 and Ser-572, recruiting the adapter protein 14-3-3, and this process leads to up-regulation of DENND3 GEF activity toward its substrate Rab. We demonstrate that phosphorylation of a specific tyrosine residue in DENND3 regulates this intramolecular interaction, suggesting that a signaling pathway involving a tyrosine kinase impinges upon the intramolecular interaction to regulate DENND3 GEF activity toward Rab
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