Baumgart Florian, Corral-Escariz María, Pérez-Gil Jesús, Rodríguez-Crespo Ignacio
Departamento de Bioquímica y Biología Molecular, Universidad Complutense, Madrid 28040, Spain.
Biochim Biophys Acta. 2010 Mar;1798(3):592-604. doi: 10.1016/j.bbamem.2010.01.002. Epub 2010 Jan 13.
Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.
最近，含有天冬氨酸 - 组氨酸 - 组氨酸 - 半胱氨酸（DHHC）基序的哺乳动物蛋白质被鉴定为一组膜相关的棕榈酰酰基转移酶（PATs）。在已知可被DHHC PATs棕榈酰化的几种蛋白质底物中，有在其羧基末端进行异戊二烯化的小GTP酶，如H-Ras或N-Ras、内皮型一氧化氮合酶（eNOS）、在其N末端进行肉豆蔻酰化的激酶，如Lck，以及许多跨膜蛋白和通道。我们的研究集中在人类基因DHHC19的产物上，它是一种假定的棕榈酰转移酶，有趣的是，在其羧基末端显示出一个保守的CaaX框。我们在此表明，DHHC19羧基末端存在的氨基酸序列能够将绿色荧光蛋白（GFP）报告基因排除在细胞核外，并将其导向核周区域。在COS7细胞中全长DHHC19的转染显示出核周分布，类似于其他棕榈酰转移酶，与反式高尔基体标记物半乳糖转移酶（Gal-T）和TGN38有强烈的共定位。我们测试了几种已知会被棕榈酰化的小GTP酶作为DHHC19可能的底物。尽管DHHC19未能增加H-Ras、N-Ras、K-Ras4A、RhoB或Rap2的棕榈酰化，但它使R-Ras的棕榈酰化增加了约两倍。与DHHC19共转染的R-Ras棕榈酰化增加伴随着与膜以及脂筏/小窝的结合增强。最后，使用野生型和R-Ras的活化GTP结合形式（G38V），我们还表明，由于DHHC19共表达导致的R-Ras棕榈酰化增加伴随着转染细胞活力的增强。
