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Abstract
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.
Highlights
Protein-tyrosine kinases of the Src family (Src PTKs)1 are cytosolic proteins, which are key elements of the signaling cascade from the surface to the interior of the cell
To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61Hck was associated with lysosomal vesicles, whereas p59Hck was found at the plasma membrane and to a low extent associated with lysosomes
We propose that bi-acylated p59Hck might transduce plasma membrane receptor signals, whereas p61Hck and the nonpalmitoylated p59Hck might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes
Summary
The wild-type Hck cDNA was a gift from N. The CTG initiation codon of Hck was either deleted or mutated to an ATG codon by polymerase chain reaction amplification of the unique N-terminal. Palmitoylation and Distinct Addressing of Hck Isoforms domain using the antisense primer Hck-AS1, with either p59-S or p61-S sense primer (see below) This introduced a XbaI and a NheI restriction site, respectively, at the 5Ј and 3Ј end of the products, which were subcloned into pBK-CMV (Stratagene, La Jolla, CA) and sequenced (Genome Express, Grenoble, France). The 114- or 176-base pair XbaI to BamHI fragments issued from p59U-Hck-GFP and p61U-Hck-GFP were used to replace the wild-type Hck N terminus yielding p59Hck or p61Hck cDNA, respectively. All oligodeoxynucleotides were synthesized by Oligo express (Grenoble, France) and were as detailed: Hck-AS1, 5Ј-CTAGCTAGCGATGATGTCCTCAGAGCC-3Ј; Hck-AS2, 5Ј-CCATCGATATCGCTAGCCGGCTGCTGTTGGTACTGGCTCTC-3Ј; Hck-S, 5Ј-CATCATCACGGAGTTCATGGCC-3Ј; p59-S, 5Ј-GCTCTAGAGCGGGCGCCCAGGATGGGGTGC-3Ј; p61-S, 5Ј-GCTCTAGAGGGGCTGCCGAGATGGGGGGGCGC-3Ј; p59C3S-S, 5Ј-GCGGCCGCCATGGGGAGCATGAAGTCCAAGTTCC-3Ј; and p59C3S-AS, 5Ј-TCCCCATGGCGGCCGCCCGCTCTAGCGGAT-3Ј
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