Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.
Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.
Wienecke
R
R
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892-4040, USA.
Maize
J C
JC
Shoarinejad
F
F
Vass
W C
WC
Reed
J
J
Bonifacino
J S
JS
Resau
J H
JH
de Gunzburg
J
J
Yeung
R S
RS
DeClue
J E
JE
D000818:Animals; D000906:Antibodies; D020126:Brefeldin A; D002478:Cells, Cultured / Q000187:drug effects; D002846:Chromatography, Affinity; D003517:Cyclopentanes / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D014018:Tissue Distribution; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D020668:rap GTP-Binding Proteins
D000906:Antibodies; D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C000624653:TSC2 protein, human; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D020126:Brefeldin A; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins