Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B.

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation

Reconsidering the Role of Cyclooxygenase Inhibition in the Chemotherapeutic Value of NO-Releasing Aspirins for Lung Cancer

Nitric oxide-releasing aspirins (NO-aspirins) are aspirin derivatives that are safer than the parent drug in the gastrointestinal context and have shown superior cytotoxic effects in several cancer models. Despite the rationale for their design, the influence of nitric oxide (NO•) on the effects of NO-aspirins has been queried. Moreover, different isomers exhibit varying antitumor activity, apparently related to their ability to release NO•. Here, we investigated the effects and mode of action of NO-aspirins in non-small-cell lung cancer (NSCLC) cells, comparing two isomers, NCX4016 and NCX4040 (-meta and -para isomers, respectively). NCX4040 was more potent in decreasing NSCLC cell viability and migration and exhibited significant synergistic effects in combination with erlotinib (an epidermal growth factor receptor inhibitor) in erlotinib-resistant cells. We also studied the relationship among the effects of NO-aspirins, NO• release, and PGE2 levels. NCX4040 released more NO• and significantly decreased PGE2 synthesis relative to NCX4016; however, NO• scavenger treatment reversed the antiproliferative effects of NCX4016, but not those of NCX4040. By contrast, misoprostol (a PGE2 receptor agonist) significantly reversed the antiproliferative effect of NCX4040, but not those of NCX4016. Furthermore, misoprostol reversed the antimigratory effects of NCX4040. Overall, these results indicate that PGE2 inhibition is important in the mode of action of NO-aspirins

Different response of APP and C99 to CQ.

<p>(A–B) H4 cells stably expressing GFP-tagged APP-F/P-D/A (A) or C99-F/P-D/A (B) were left untreated or treated for 16 h either with 1 µM DAPT, 100 µM CQ, or with a combination of 1 µM DAPT and 100 µM CQ. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left. (C) Densitometric quantification of the levels of APP and C99 shown in A and B. Bars represent the mean ± SD (APP n = 7; C99 n = 6). *<i>P</i><0.05. (D) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged APP-F/P-D/A or C99-F/P-D/A left untreated (Control) or treated with 100 µM CQ for 16 h. Bar, 10 µm.</p

Accumulation of C99 in response to MG132, CQ and lack of its cytosolic tyrosine residues.

<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its three tyrosine residues (bold underline). (B) Immunoblot analysis of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-3Y/A-F/P-D/A (C99-3Y/A). Cells were left untreated or treated with 1 µM DAPT for 16 h and subsequently analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin was used as loading control. The positions of molecular mass markers are indicated on the left. (C) H4 cells stably expressing C99-3Y/A were left untreated or treated for 16 h either with 1 µM MG132, 100 µM CQ, or with a combination of 1 µM MG132 and 100 µM CQ. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of C99-3Y/A left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bars represent the mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p

Degradation of C99 after redistribution to the endoplasmic reticulum requires polyubiquitination of its cytosolic lysine residues.

<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its five lysine residues (bold underline). (B–D) H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) were processed as follows: (B) transfected with HA-tagged ubiquitin and left untreated or treated with 1 µM MG132 for 4 h, and after denaturation soluble extracts immunoprecipitated with anti-GFP antibody; (C) incubated with 150 µg/ml CHX and 40 µg/ml chloramphenicol for 0–30 min; or (D) pretreated with 5 µg/ml BFA for 1 h before further incubation with BFA and the combination of CHX and chloramphenicol for 0–30 min. Proteins were analyzed by immunoblot with HRP-conjugated anti-HA antibody (B; C99-Ub-HA), or anti-GFP antibody (B–D). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (E–F) Confocal fluorescence microscopy of cells stably expressing GFP-tagged C99 (E) or C99-5K/R (F) left untreated (Control) or treated with 5 µg/ml BFA for 1 h. Bars, 10 µm.</p

Accumulation of C99 at the cell surface in response to MG132 and CQ.

<p>(A) H4 cells stably expressing GFP-tagged C99-F/P-D/A were left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (B) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged C99-F/P-D/A treated for 16 h either with 1 µM MG132 or with a combination of 1 µM MG132 and 100 µM CQ. Bar, 10 µm. (C) Densitometric quantification of the levels of C99 and AICDγ shown in A. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05; **<i>P</i><0.01.</p

C99 is proteolytically cleaved in different sites.

<p>(A) Schematic representation of GFP-tagged C99, C83 and C31 indicating their topological domains, and the position of the Aβ peptide, the p3 peptide, the proteolytic cleavage sites (α, γ and caspase), and the AICDγ fragment. (B) H4 cells transiently expressing wild-type C99-GFP (WT) or C99-GFP with either the D87A mutation, the F38P mutation, or both (F/P-D/A), were left untreated or treated with 1 µM DAPT for 16 h. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left.</p

Intracellular localization and proteolytic processing of C99.

<p>(A) Schematic representation of GFP-tagged APP and C99 indicating their topological domains and the position of the HA tag, the Aβ peptide, the proteolytic cleavage sites (α, β and γ), and the AICDγ fragment. (B) Fluorescence microscopy analysis of H4 human neuroglioma cells transiently expressing APP-GFP or C99-GFP. Bar, 10 µm. (C–E) H4 cells transiently expressing C99-GFP were left untreated or treated for 16 h with 1 µM DAPT, labeled for 4 hr at 20°C with 1 mCi/ml [³⁵S]-methionine-cysteine, and chased at 37°C for the indicated times. C99 and Aβ species were immunoprecipitated from cell lysates with anti-GFP antibody (C), or from the culture medium with 6E10 antibody (E), respectively. Proteins were analyzed on 10%–20% Tricine gels and fluorography. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of the levels of C99, C83, and AICDγ shown in C.</p

Proposed processing and turnover routes of C99.

<p>(A) (<i>i</i>) A small fraction of newly-synthesized APP in the endoplasmic reticulum (ER) can be a substrate of BACE1 that generates C99. Ubiquitinated (Ub) C99 can be a substrate of the endoplasmic reticulum-associated protein degradation (ERAD) pathway to ultimately be degraded by the proteasome. (<i>ii</i>) En route through the secretory pathway, a fraction of APP at the Golgi apparatus can also be a substrate of BACE1 that generates C99, which subsequently can be a substrate of γ-secretase (γ-sec) activity that generates Aβ peptides and cytosolic AICDγ, a proteolytic processing that can be inhibited by DAPT. (<i>iii</i>) Finally, within endo/lysosomal compartments APP can be degraded by acid hydrolases. (B) (<i>i</i>) Upon MG132 inhibition, ubiquitinated C99 accumulates within the ER. Ubiquitinated C99 can exit the ER and reach the Golgi apparatus. (<i>ii</i>) Both ubiquitinated C99 and C99 generated from APP can be cleaved at the Golgi apparatus by γ-secretase activity. Upon Brefeldin A (BFA) treatment, C99 can be relocated from the Golgi apparatus to the ER where it can be also cleaved by γ-secretase activity. (<i>iii</i>) Both APP and the excess of C99 can be degraded by acid hydrolases. (C) (<i>i</i>) Upon MG132 treatment, and (<i>ii</i>) the generation of an excess of C99 at the Golgi apparatus, (<i>iii</i>) chloroquine (CQ) treatment results in accumulation of both APP and C99 within endo/lysosomal compartments. For simplicity, other APP metabolites, such as sAPPβ, which is the other product of BACE1 activity on APP, or the C31 fragment, are not depicted.</p

C99 is degraded after redistribution to the endoplasmic reticulum.

<p>H4 cells stably expressing GFP-tagged C99-F/P-D/A were treated as follows: (A) with increasing concentrations of MG132 for 4 h; (B) left untreated or treated either with 1 µM DAPT for 16 h, 1 µM MG132 for 4 h, or 1 µM DAPT for 12 h followed by a combination of 1 µM DAPT and 1 µM MG132 for 4 h; (C) left untreated or treated for 4 h either with 5 µg/ml BFA, 1 µM MG132, or a combination of 1 µM MG132 and 5 µg/ml BFA; or (D) pretreated with 5 µg/ml BFA without or with 1 µM MG132 for 4 h followed by CHX-chase for 0–60 min without or with 1 µM MG132. (E) H4 cells stably expressing GFP-tagged APP-F/P-D/A were left untreated or treated for 4 h either with 5 µg/ml BFA, or a combination of 5 µg/ml BFA and 1 µM MG132. Cellular extracts were analyzed by immunoblot with anti-GFP antibody (A–E), or WO2 monoclonal antibody to detect C99 in cells expressing GFP-tagged APP-F/P-D/A (E). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (F) Densitometric quantification of the levels of C99 shown in E. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05.</p