Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome

Abstract

Background: Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.

Results: We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCFSkp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins in vivo. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.

Conclusion: SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.

Figure 1

SAP130 is a CSN1 interacting protein. (A) Diagram of bait HT-FS1-NTD. From the N-terminus, this protein contains a His-tag (6 × HIS), Tobacco Etch Virus protease recognition and cleavage site (TEV site) and Flag tagged CSN1-NTD (1–196 aa). (B) Coomassie Blue staining gel showing the input NIH3T3 cell lysate and proteins eluted after TEV protease cleavage. Identity of corresponding proteins is labeled. (C) SAP130-myc was co-transfected in HeLa cells with empty vector (lane 1) or Flag-tagged CSN subunits as indicated. Total cell extract and the Flag immunoprecipitated (IP) proteins were probed with anti-myc antibody. (D) CSN1 was immunoprecipitated from DRB treated NIH3T3 nuclear extract. Endogenous SAP130 was enriched in CSN1 IP compared to pre-immune serum (pre-im). (E) HA-SAP130 was in vitro translated and S³⁵-Met labeled in rabbit reticulocyte lysate. The samples were incubated with recombinant GST or GST-CSN1 proteins (1 μg each) for 30 min. Following GST pull-down, samples were analyzed by autoradiogram (upper panel) or anti-GST immunoblotting (lower panel). (F) HA-CPSF160 was transiently co-transfected in HeLa cells with empty vector (lane 1) or Flag-tagged CSN subunits. Total cell extract and the Flag immunoprecipitated proteins were probed with anti-HA antibody.

Figure 2

SAP130 interacts with Nedd8 modified cullins. (A) Whole cell extracts (lysate) and anti-HA immunoprecipitate (IP HA) from HEK293 cells transiently transfected with empty vector (-) or HA-SAP130 (+) were analyzed by immunoblotting using the antibodies against cullins or SAP155, a component of SF3b RNA-splicing complex. (B) Rabbit reticulocyte lysate (RRL) containing in vitro translated HA-SAP130 were incubated with recombinant GST-Nedd8, GST-Ub or GST (1 μg each). HA IP samples were analyzed by anti-Cul2 and anti-GST blots and autoradiogram (S³⁵). (C) HA-SAP130 was in vitro translated in RRL supplemented with GST or GST-Nedd8 (1 μg each). Anti-GST blot shows that free GST-Nedd8, like GST alone, was not associated with HA-SAP130. (D) HeLa Cells were transfected with siRNA reagent against CAND1 or laminin-A control. Input lysate or HA IP samples were blotted with anti-CAND1 or Cul2 antibodies. Note that there was weak non-specific binding of un-neddylated Cul2 to HA beads (lane 1).

Figure 3

SAP130 forms tertiary complexes with CRL substrate adaptors and is associated with E3 activity. (A) HEK293 cells were transfected with HA-VHL or Flag-SAP130. Anti-HA and anti-Flag IP samples were blotted with indicated antibodies. An asterisk (*) indicates a non-specific band in Flag IP. (B) HA-SAP130 was expressed in HEK293 cells. Anti-HA IP samples were probed with indicated antibodies. (C) Flag immunocomplex isolated from HEK293 cells expressing empty vector, Flag-SAP130 or Flag-CPSF160 were incubated with N-terminal biotin-labeled ubiquitin, E1 and E2 (UbcH5b) as indicated. Polyubiquitination was determined by the detection of biotin-Ub.

Figure 4

Interaction of SAP130 with HA-Cul1. (A) A diagram indicating the Cul1 truncation constructs and the interaction results. (B) HA-Cul1 and its truncations HA-Cul1N428, HA-Cul1C450 and HA-Cul1C280 were transiently expressed in HEK293 cells. The HA-IP samples were blotted with indicated antibodies. (C) HA-Cul1 and its truncations as indicated were transfected into Flag-SAP130 stable cells. Flag IP was performed and samples were probed with anti-HA antibody. (D) HeLa cells were transfected with HA-Cul1 or vector. Cell extracts were incubated with anti-CSN2 antibody or pre-immune serum for 15 min at room temperature and then blotted for Cul1 or HA. Arrowheads indicate endogenous Cul1 and the lines on the right indicate overexpressed HA-Cul1.

Figure 5

SAP130 binds to Cul2 CTD. (A) HA-Cul2 and its truncations HA-Cul2N483 and HA-Cul2C261 were transiently transfected in HEK293 cells. The HA-IP samples were blotted with antibodies against HA, Nedd8, Elongin B or CAND1. Nedd8 modified and un-modified Cul2 proteins are indicated. (B) HA-Cul2 and its truncations as indicated were transfected into Flag-SAP130 stable cells (upper panel) or HEK293 cells along with Flag-CSN1 (bottom panel). Flag IP was performed and samples were blotted using anti-HA antibody. (C) A diagram indicating the Cul2 truncation constructs and the interaction results from (A) and (B).

Figure 6

CSN1 inhibits SAP130-Cul2 interaction and SAP130 associated E3 activity. (A) In vitro translated HA-SAP130 in RRL was incubated with GST-Nedd8 (1 μg) and GST-CSN1 (0.5 μg or 1.0 μg) or GST (1.0 μg). The RRL input and HA-IP were analyzed by anti-Cul2 blot. (B) Whole cell extract from Flag-SAP130 stable cells were either left untreated or pretreated with GST (2 μg), GST-CSN1 (1 μg and 2 μg) or purified CSN complex from porcine spleen (CSN com). Flag immunocomplex was subsequently isolated and used for in vitro E3 activity assay. Polyubiquitin chain was detected by anti-ubiquitin blot. Equal amount of Flag-SAP130 immunocomplex was confirmed by anti-Flag blot. Cul2 blot of the pretreated extract showed reduced neddylation after incubation with CSN complex but not GST-CSN1.