Identification of in Vitro Phosphorylation Sites in the Growth Cone Protein SCG10
EFFECT OF PHOSPHORYLATION SITE MUTANTS ON MICROTUBULE-DESTABILIZING ACTIVITY*
- Bruno Antonsson‡§,
- Daniel B. Kassel¶‖,
- Gilbert Di Paolo**,
- Robert Lutjens**,
- Beat M. Riederer** and
- Gabriele Grenningloh**‡
- From the ‡Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 1228 Plan-les-Ouates, Switzerland, ¶Glaxo Inc., Research Triangle Park, North Carolina 27709, and **Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, 1005 Lausanne, Switzerland
Abstract
SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitrophosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.
Footnotes
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↵* This work was supported in part by the National Science Foundation of Switzerland, Grants 31-43137.95 (to B. M. R.) and 3100-050948.97 (to G.G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Present address: Serono Pharmaceutical Research Institute S. A., 1228 Plan-les-Ouates, Switzerland.
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↵‖ Present address: CombiChem, Inc., San Diego, CA 92121.
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↵‡ To whom correspondences should be addressed: IBCM, Université de Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland. Tel.: 41-21-692 5100; Fax: 41-21-692 5105.
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↵1 The abbreviations used are: MT, microtubule; PKA, cAMP-dependent protein kinase; MAP, mitogen-activated protein; CDK (p34cdc2 and CDK5/p25), cyclin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; MS/MS tandem MS; LC, liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.
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- Received October 22, 1997.
- Revision received January 26, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
