EPI64 Protein Functions as a Physiological GTPase-activating Protein for Rab27 Protein and Regulates Amylase Release in Rat Parotid Acinar Cells

Abstract

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/ Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.Small GTPase Rabs are believed to play important roles in intracellular membrane trafficking (reviewed in Refs. 1-4). More than 60 distinct Rab isoforms have been identified in humans and mice, and these proteins form a large subfamily of the small GTPase superfamily (5-7). Rab activity is regulated by the GDP/GTP cycle (1-4, 8), with GTP-Rab and GDP-Rab representing active and inactive forms, respectively. Distinct individual Rabs interact with a specific effector/regulator, and these complexes regulate different steps of membrane trafficking in cells, and they function as either an accelerator or a brake (9, 10). GTP bound to a particular Rab is hydrolyzed by intrinsic GTPase activity, and this activity is enhanced by a specific GTPase-activating protein (GAP) 3 (11). The TBC (Tre-2/Bub2/Cdc16) domain is a conserved protein motif with ϳ200 amino acids and is known to be present in a variety of molecules in eukaryotic organisms (12). Because the TBC domain of yeast, Gyps (GAP for Ypt proteins), has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (referred to as TBC proteins hereafter) in other species are also expected to function as specific Rab-GAPs. Humans and mice each have more than 40 TBC proteins, and substantial evidence has accumulated recently indicating that some...

PI3K regulates endocytosis after insulin secretion via signaling crosstalk between Arf6 and Rab27a

“…EPI64 also functions as a Rab27a GAP through its Tre-Bud-Cdc16 (TBC) domain Imai et al, 2011). We have recently shown that glucose induces the conversion of GTP-bound Rab27a into its GDP-bound form through EPI64 in pancreatic β-cells (Kimura et al, 2010b).…”

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells

“…However, Rab3GAP was identified as specific for Rab3 subfamily members (44). A recent study has demonstrated that EPI64/ TBC1D10A can serve as a physiological GAP for Rab27A in parotid acinar cells and regulate amylase release (45). We verified the expression of EPI64 and EPI64B in isolated pancreatic acini by RT-PCR and EPI64B by Western blotting and then made epitope-tagged EPI64 and EPI64B adenoviruses.…”

“…A recently published study showed that inhibition of EPI64, either by antibody or antisense locked nucleic acid, reduced amylase release from parotid acinar cells (45). It has also been reported that in pancreatic acini, knockout of Slp1, a downstream effector of Rab27B, increased both the numbers of zymogen granules and amylase secretion in fasted mice (50).…”

“…In addition to Rab27A, Rab35 has also been shown to be a target small G protein of EPI64B (46). Due to different functions and limited involvement of Rab35 in secretion in acinar cells (45), EPI64B is likely to mainly act as Rab27B GAP at the final secretory steps in the pancreatic acinar cells. Taken together, due to the abundance on the zymogen granules and involvement in the exocytosis, Rab27B should be the primary target of EPI64B in pancreatic acinar cells.…”

Rab27

“…In addition, a homologue of EPI64, EPI64B also exhibited Rab27A GAP activity in vitro. EPI64 is a physiological GAP for Rab27 in rat parotid acinar cells and down-regulation of EPI64 caused a reduction in the amount of amylase release (33). A recent study showed that EPI64 also functions as Rab27A specific GAP in a lung adenocarcinoma cell line and regulates exosome secretion.…”