Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes.
Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61(Hck) has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59(Hck) N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61(Hck) was associated with lysosomal vesicles, whereas p59(Hck) was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59(Hck) redirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59(Hck) with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59(Hck) mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59(Hck) might transduce plasma membrane receptor signals, whereas p61(Hck) and the nonpalmitoylated p59(Hck) might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.
Carréno
S
S
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5089, 31077 Toulouse, France.
Gouze
M E
ME
Schaak
S
S
Emorine
L J
LJ
Maridonneau-Parini
I
I
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019308:Palmitic Acid; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck