Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box.
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.
Baumgart
Florian
F
Departamento de Bioquímica y Biología Molecular, Universidad Complutense, Madrid 28040, Spain.
Corral-Escariz
María
M
Pérez-Gil
Jesús
J
Rodríguez-Crespo
Ignacio
I
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D021941:Caveolae / Q000201:enzymology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D054878:Lipoylation; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D021381:Protein Transport; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000217:Acyltransferases; C548831:ZDHHC19 protein, human; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins