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.
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August 2002
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Research Article|
August 15 2002
Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases
Marco MUDA;
Marco MUDA
∗Serono Reproductive Biology Institute, Inc., Randolph, MA 02368, U.S.A.
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Carolyn A. WORBY;
Carolyn A. WORBY
†The Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, U.S.A.
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Nancy SIMONSON-LEFF;
Nancy SIMONSON-LEFF
†The Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, U.S.A.
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James C. CLEMENS;
James C. CLEMENS
‡Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA School of Medicine, Los Angeles, CA 90095, U.S.A.
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Jack E. DIXON
Jack E. DIXON
1
†The Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, U.S.A.
1To whom correspondence should be addressed (e-mail jedixon@umich.edu).
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Publisher: Portland Press Ltd
Received:
February 21 2002
Revision Received:
May 02 2002
Accepted:
May 16 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (1): 73–77.
Article history
Received:
February 21 2002
Revision Received:
May 02 2002
Accepted:
May 16 2002
Citation
Marco MUDA, Carolyn A. WORBY, Nancy SIMONSON-LEFF, James C. CLEMENS, Jack E. DIXON; Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J 15 August 2002; 366 (1): 73–77. doi: https://doi.org/10.1042/bj20020298
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