Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes

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

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

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

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

The cytosolic and membrane pools of GOLPH3 are differentially modified in different human breast cell lines.

<p>Samples (30 μg of proteins) of rat liver cytosol (<i>Cyt</i>), rat liver Golgi membranes, and of cytosolic (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase (<i>CIAP</i>) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point (<i>pI</i>) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.</p

The cytosol of different human breast cell lines affects differently the avidity of GOLPH3 for phosphatidylinositol 4-phosphate.

<p>(A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 (<i>GOLPH3</i>) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. <i>LysoPtdA</i>, lysophosphatidic acid; <i>LysoPtdCho</i>, lysophosphatidylcholine; <i>PtdIns</i>, phosphatidylinositol; <i>PtdIns(3)P</i>, phosphatidylinositol 3-phosphate; <i>PtdIns(4)P</i>, phosphatidylinositol 4-phosphate; <i>PtdIns(5)P</i>, phosphatidylinositol 5-phosphate; <i>PtdEth</i>, phosphatidylethanolamine; <i>PtdCho</i>, phosphatidylcholine; <i>S1P</i>, sphingosine 1-phosphate; <i>PtdIns(3</i>,<i>4)P</i>_<i>2</i>, phosphatidylinositol 3,4-bisphosphate; <i>PtdIns(3</i>,<i>5)P</i>_<i>2</i>, phosphatidylinositol 3,5-bisphosphate; <i>PtdIns(4</i>,<i>5)P</i>_<i>2</i>, phosphatidylinositol 4,5-bisphosphate; <i>PtdIns(3</i>,<i>4</i>,<i>5)P</i>_<i>3</i>, phosphatidylinositol 3,4,5-trisphosphate; <i>PtdA</i>, phosphatidic acid; <i>PtdSer</i>, phosphatidylserine; <i>Blank</i>, no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * <i>P</i> < 0.05; *** <i>P</i> < 0.001.</p