Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.
Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.
Muda
Marco
M
Serono Reproductive Biology Institute, Inc., Randolph, MA 02368, U.S.A.
Worby
Carolyn A
CA
Simonson-Leff
Nancy
N
Clemens
James C
JC
Dixon
Jack E
JE
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001369:Axons / Q000378:metabolism; D015153:Blotting, Western; D015815:Cell Adhesion Molecules; D004330:Drosophila; D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D029721:Drosophila Proteins; C491736:Dscam1 protein, Drosophila; D009419:Nerve Tissue Proteins; D011506:Proteins; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C099753:dock protein, Drosophila; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C107962:Ptp61F protein, Drosophila