GOLPH3: a Golgi phosphatidylinositol(4)phosphate effector that directs vesicle trafficking and drives cancer

GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4)P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans-Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer. GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4)P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans-Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer. Cancer involves wholesale production of cellular biomass, and thus we might predict that every part of the cell, and every organellar regulatory pathway, is likely to be involved. Indeed, many organelles, including mitochondria, lysosomes, and the endoplasmic reticulum (ER), have well-established roles in promoting cancer initiation and/or progression (1Papaioannou A. Chevet E. Driving cancer tumorigenesis and metastasis through UPR signaling.Curr. Top. Microbiol. Immunol. 2018; 414: 159-192PubMed Google Scholar, 2Pavlova N.N. Thompson C.B. 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These observations suggest that the GOLPH3 complex applies a stretching force to the Golgi that is responsible for its characteristic appearance. Although the morphological effects are striking, it is unlikely that this is the purpose of the GOLPH3 complex (41Buschman M.D. Xing M. Field S.J. The GOLPH3 pathway regulates Golgi shape and function and is activated by DNA damage.Front. Neurosci. 2015; 9: 362Crossref PubMed Scopus (29) Google Scholar). For example, the GOLPH3 complex is conserved and functional in organisms and cell types with dramatically differing Golgi morphology. Rather, we find that the GOLPH3 complex plays a critical role in the exit of vesicles from the Golgi for trafficking to the plasma membrane, and the effect on Golgi shape is a consequence of the mechanism of vesicle budding. Indeed, live imaging reveals that the majority of PtdIns(4)P-positive, cargo-positive vesicles that exit from the Golgi move in a direction parallel to the tensile force, as indicated by the shape of the Golgi (11Dippold H.C. Ng M.M. Farber-Katz S.E. Lee S-K. Kerr M.L. Peterman M.C. Sim R. Wiharto P.A. Galbraith K.A. Madhavarapu S. et al.GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.Cell. 2009; 139: 337-351Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Interference with the GOLPH3 complex [e.g., by depletion of PtdIns(4)P, GOLPH3, MYO18A, or F-actin] results in a dramatic reduction in the exit of these vesicles from the trans-Golgi (11Dippold H.C. Ng M.M. Farber-Katz S.E. Lee S-K. Kerr M.L. Peterman M.C. Sim R. Wiharto P.A. Galbraith K.A. Madhavarapu S. et al.GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.Cell. 2009; 139: 337-351Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Accordingly, interference with the GOLPH3 complex dramatically reduces Golgi-to-plasma membrane trafficking, as observed by a variety of experiments. Global protein secretion, as measured by 35S-Met pulse/chase and assessment of the appearance of label incorporated into proteins in the media, is inhibited >80% by knockdown of GOLPH3 or MYO18A, similar to the effect of the Golgi poison Brefeldin A (12Ng M.M. Dippold H.C. Buschman M.D. Noakes C.J. Field S.J. GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.Mol. Biol. Cell. 2013; 24: 796-808Crossref PubMed Scopus (55) Google Scholar). Trafficking of VSVG to the plasma membrane is inhibited >50%, with accumulation at the Golgi, by knockdown of GOLPH3 or MYO18A (11Dippold H.C. Ng M.M. Farber-Katz S.E. Lee S-K. Kerr M.L. Peterman M.C. Sim R. Wiharto P.A. Galbraith K.A. Madhavarapu S. et al.GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.Cell. 2009; 139: 337-351Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 42Xing M. Peterman M.C. Davis R.L. Oegema K. Shiau A.K. Field S.J. GOLPH3 drives cell migration by promoting Golgi reorientation and directional trafficking to the leading edge.Mol. Biol. Cell. 2016; 27: 3828-3840Crossref PubMed Scopus (43) Google Scholar). Secretion of hepatitis C virus from infected cells is inhibited >80%, with intracellular accumulation of intact, otherwise fully functional viral particles, upon knockdown of GOLPH3 or MYO18A (43Bishé B. Syed G.H. Field S.J. Siddiqui A. Role of phosphatidylinositol 4-phosphate (PI4P) and its binding protein GOLPH3 in hepatitis C virus secretion.J. Biol. 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The discovery of the GOLPH3 complex has provided a framework for understanding regulation of the Golgi. Based on our assumption that the GOLPH3 complex is likely to be regulated, we mapped phosphorylation sites in GOLPH3 (44Farber-Katz S.E. Dippold H.C. Buschman M.D. Peterman M.C. Xing M. Noakes C.J. Tat J. Ng M.M. Rahajeng J. Cowan D.M. et al.DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.Cell. 2014; 156: 413-427Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). One of the sites, Thr143, we found was phosphorylated by the DNA damage-activated protein kinase, DNA-PK. This led to our discovery of the Golgi DNA damage response, a common feature of the cellular response to DNA damage in mammals, whereby the Golgi ribbon fragments into small vesicles that disperse throughout the cytoplasm (44Farber-Katz S.E. Dippold H.C. Buschman M.D. Peterman M.C. Xing M. Noakes C.J. Tat J. Ng M.M. Rahajeng J. Cowan D.M. et al.DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.Cell. 2014; 156: 413-427Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, we found that phosphorylation of GOLPH3 on Thr143 by DNA-PK in response to DNA damage enhances the interaction with MYO18A, leading to increased vesiculation of the Golgi, resulting in Golgi fragmentation. Ultimately, this hyperactivation of the GOLPH3 complex is required for normal cell survival following DNA damage (44Farber-Katz S.E. Dippold H.C. Buschman M.D. Peterman M.C. Xing M. Noakes C.J. Tat J. Ng M.M. Rahajeng J. Cowan D.M. et al.DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.Cell. 2014; 156: 413-427Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Another example of regulation of the Golgi through the GOLPH3 complex involves growth factor stimulation of an increase in PtdIns(4)P levels at the Golgi (45Blagoveshchenskaya A. Cheong F.Y. Rohde H.M. Glover G. Knödler A. Nicolson T. Boehmelt G. Mayinger P. Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1.J. Cell Biol. 2008; 180: 803-812Crossref PubMed Scopus (117) Google Scholar, 46Bajaj Pahuja K. Wang J. Blagoveshchenskaya A. Lim L. Madhusudhan M.S. Mayinger P. Schekman R. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2015; 112: E3199-E3206Crossref PubMed Scopus (27) Google Scholar). Growth factor signaling was found to drive trafficking of the SAC1 PtdIns(4)P-phosphatase from the Golgi to the ER, allowing for elevated levels of PtdIns(4)P at the Golgi. Withdrawal of growth factors leads to an increase in SAC1 at the Golgi and a concomitant decrease in PtdIns(4)P levels at the Golgi, associated with compaction of the Golgi ribbon and reduced Golgi-to-plasma membrane trafficking, the hallmarks of inhibition of the GOLPH3 complex. The mechanism of regulation involves growth factor-stimulated activation of p38/MAPK to regulate SAC1's interaction with the COPI retrograde trafficking machinery (45Blagoveshchenskaya A. Cheong F.Y. Rohde H.M. Glover G. Knödler A. Nicolson T. Boehmelt G. Mayinger P. Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1.J. Cell Biol. 2008; 180: 803-812Crossref PubMed Scopus (117) Google Scholar, 46Bajaj Pahuja K. Wang J. Blagoveshchenskaya A. Lim L. Madhusudhan M.S. Mayinger P. Schekman R. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2015; 112: E3199-E3206Crossref PubMed Scopus (27) Google Scholar). The activity of the GOLPH3 complex is also differentially regulated during cell-type specification by the selective expression of GOLPH3L in highly secretory cells (12Ng M.M. Dippold H.C. Buschman M.D. Noakes C.J. Field S.J. GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.Mol. Biol. Cell. 2013; 24: 796-808Crossref PubMed Scopus (55) Google Scholar). GOLPH3L is a paralog of GOLPH3 that, like GOLPH3, binds to PtdIns(4)P and localizes to the Golgi. However, unlike GOLPH3, GOLPH3L does not interact with MYO18A. As such, GOLPH3L acts as an endogenous dominant-negative inhibitor of the GOLPH3 complex, with opposite effects on the Golgi compared with GOLPH3 (12Ng M.M. Dippold H.C. Buschman M.D. Noakes C.J. Field S.J. GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.Mol. Biol. Cell. 2013; 24: 796-808Crossref PubMed Scopus (55) Google Scholar, 38Xie Z. Hur S.K. Zhao L. Abrams C.S. Bankaitis V.A. A Golgi lipid signaling pathway controls apical Golgi distribution and cell polarity during neurogenesis.Dev. Cell. 2018; 44: 725-740.e4Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The data indicate that GOLPH3L acts as a throttle in highly secretory cells. These examples provide a picture of the GOLPH3 complex as a hub for convergent signals for regulation of the Golgi. The old picture of a constitutive Golgi, with little or no regulation, was not biologically plausible. The GOLPH3 complex has provided a conceptual framework to explore and understand regulation of the Golgi, which is likely to be rich and to involve many intracellular and extracellular signaling pathways. Unbiased cancer genome studies have independently identified GOLPH3, MYO18A, and PITPNC1 as drivers of common human cancers. GOLPH3 was found in a genome-wide search for genes that are frequently amplified in human cancers (47Scott K.L. Kabbarah O. Liang M-C. Ivanova E. Anagnostou V. Wu J. Dhakal S. Wu M. Chen S. Feinberg T. et al.GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer.Nature. 2009; 459: 1085-1090Crossref PubMed Scopus (288) Google Scholar). A minimal region of chromosome 5p13, encompassing four genes, was found to be common to all of the amplifications. Of these, GOLPH3 alone was found to have expression levels that correlated with copy number, and its knockdown was found to revert oncogenic transformation in cell culture. The GOLPH3 gene was found to be amplified in 56% of lung carcinomas, 37% of prostate carcinomas, 32% of breast carcinomas, 33% of pancreatic carcinomas, 24% of colon carcinomas, and 37% of ovarian carcinomas. The Cancer Genome Atlas (TCGA) reports rates of amplification of the GOLPH3 gene that are somewhat lower, although still significant, e.g., in 15% of lung squamous cell carcinomas and 10% of lung adenocarcinomas (48Cancer Genome Atlas Research Network Comprehensive genomic characterization of squamous cell lung cancers.Nature. 2012; 489: 519-525Crossref PubMed Scopus (2925) Google Scholar, 49Cancer Genome Atlas Research Network Comprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3557) Google Scholar). In cell culture and xenog