The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression

Abstract

The class III phosphatidylinositol 3-kinase (PI3KC3) plays a central role in autophagy. Rubicon, a RUN domain-containing protein, is newly identified as a PI3KC3 subunit through its association with Beclin 1. Rubicon serves as a negative regulator of PI3KC3 and autophagosome maturation. The molecular mechanism underlying the PI3KC3 and autophagy inhibition by Rubicon is largely unknown. Here, we demonstrate that Rubicon interacts with the PI3KC3 catalytic subunit hVps34 via its RUN domain. The RUN domain contributes to the efficient inhibition of PI3KC3 lipid kinase activity by Rubicon. Furthermore, a Rubicon RUN domain deletion mutant fails to complement the autophagy deficiency in Rubicon-depleted cells. Hence, these results reveal a critical role of the Rubicon RUN domain in PI3KC3 and autophagy regulation.

FIGURE 1.

Rubicon is part of the PI3KC3 complex with UVRAG. A, cell extracts from U2OS cells expressing ZZ-Rubicon-FLAG at physiological levels or vector alone (control (Con)) were collected and purified sequentially through IgG beads and FLAG M2 beads. The resulting complex was resolved by 4–12% gradient SDS-PAGE and silver staining and analyzed by mass spectrometry. The identified proteins are indicated. M, molecular mass markers; TAP, tandem affinity purification. B, U2OS cell extracts were immunoprecipitated (IP) with anti-hVps34 or anti-Rubicon antibody, and the resulting immunoprecipitates were analyzed by Western blotting (immunoblotting (IB)).

FIGURE 2.

Rubicon physically interacts with UVRAG and hVps34. A, full-length FLAG-tagged (F) or FLAG-His-double-tagged (FH) recombinant hVps34, Beclin 1, and Barkor/Atg14(L). Rubicon and UVRAG were purified from baculovirus-infected insect cells or transfected HEK293T cells. B, Rubicon interacts directly with UVRAG and hVps34 in the purified system. Purified Rubicon-FLAG-His₆ or Barkor/Atg14(L)-FLAG-His₆ was first bound to Ni-NTA beads. Purified recombinant proteins for hVps34, UVRAG, and Beclin 1 were then allowed to pass through the Ni-NTA beads (even-numbered lanes). Bound proteins were detected by Western blotting using anti-FLAG antibody. The empty Ni-NTA beads were used as controls (odd-numbered lanes).

FIGURE 3.

Rubicon binds to hVps34 via the RUN domain. A, in Rubicon, the three highlighted domains are the RUN domain, CCD, and FYVE-like domain. FLAG-tagged wild-type Rubicon and deletion mutants, including deletions of the RUN domain, CCD, or FYVE-like domain from full-length Rubicon, as well as three large polypeptide fragments containing the RUN domain, CCD, or FYVE-like domain were coexpressed with Myc-UVRAG in HEK293T cells. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibody. The bound proteins (B) or the 5% input extract was detected by immunoblotting (IB) using the antibodies indicated. WCL, whole cell lysate. B, the N terminus of Rubicon (aa 1–300) was further divided into three FLAG-tagged fragments containing aa 1–48, 49–180 (RUN domain), and 181–300, respectively. These deletion constructs were coexpressed with Myc-hVps34 in HEK293T cells. The cell extracts were immunoprecipitated with FLAG M2 beads, followed by Western blotting for Myc-hVps34 and FLAG-tagged Rubicon fragments.

FIGURE 4.

The RUN domain of Rubicon contributes to efficient inhibition of hVps34 lipid kinase activity. A, vector alone, the N-terminal region (aa 1–300), and the C-terminal region (aa 181–972) of Rubicon were coexpressed with GFP-2×FYVE in HEK293T cells. GFP-2×FYVE puncta were observed under a fluorescence microscope. 3-MA, 3-methyladenine. B, quantitative analysis (summarized from 100 cells) of GFP-2×FYVE puncta in the cells described in A. C, FLAG-tagged hVps34 was coexpressed in HEK293T cells with one of the following Myc-tagged proteins: Beclin 1, Barkor/Atg14(L), UVRAG, Rubicon, or Rubicon-ΔRUN. D, cell lysates from the cells described in C were immunoprecipitated with M2 beads and assayed for PI3K kinase activity in a TLC assay. The phosphorylation product [³²P]PI(3)P was separated by TLC and further visualized using a Fuji phosphorimaging scanner.

FIGURE 5.

The Run domain is important for autophagy suppression by Rubicon. A, Rubicon shRNA-non-induced U2OS cells (4i DOX−) and doxycycline-induced RNAi-depleted cells (4i DOX+) were observed under a transmission electron microscope. Autophagic vacuoles are indicated by arrows. Clone 4 expressing shRNA against Rubicon (Rubicon 4i) was used in this study. Scale bars = 2 μm. B, Parental U2OS cells (U2OS cells expressing the tet repressor (U2OS-TR)), Rubicon shRNA-non-induced U2OS cells (4i DOX−), RNAi-depleted cells (4i DOX+), and Rubicon-depleted cells complemented with vector alone, FLAG-tagged wild-type Rubicon, the Rubicon C-terminal deletion (ΔCT; aa 800–972), and the Rubicon RUN domain deletion (ΔRUN; aa 1–180) were observed under a transmission electron microscope. Autophagic vacuoles are marked by arrows. C, quantitative results (summarized from 50 cells/cell line) of the autophagic vacuoles (AVs) described in B. D, Rubicon was detected by anti-FLAG M2 antibody; p62 and tubulin were detected with the respective antibodies by immunoblotting of the cells described in B.