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.

Protein kinase A activity is necessary for fission and fusion of Golgi to endoplasmic reticulum retrograde tubules.

It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity.