TY - JOUR
T1 - Rab35–GEFs, DENND1A and folliculin differentially regulate podocalyxin trafficking in two- And three-dimensional epithelial cell cultures
AU - Kinoshita, Riko
AU - Homma, Yuta
AU - Fukuda, Mitsunori
N1 - Funding Information:
This work was supported in part by Grant-in-Aid for Young Scientists 18K14692 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Y. H.); Grant-in-Aid for Scientific Research (B) 19H03220 from MEXT (to M. F.); and Japan Science and Technology Agency (JST) CREST Grant JPMJCR17H4 (to M. F.). The authors declare that they have no conflicts of interest with the contents of this article.
Publisher Copyright:
© 2020 Kinoshita et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/3/13
Y1 - 2020/3/13
N2 - Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin–Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain– containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
AB - Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin–Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain– containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
UR - http://www.scopus.com/inward/record.url?scp=85081951933&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85081951933&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.011646
DO - 10.1074/jbc.RA119.011646
M3 - Article
C2 - 31992598
AN - SCOPUS:85081951933
VL - 295
SP - 3652
EP - 3663
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 11
ER -