Identification of in vitro phosphorylation sites in the growth cone protein SCG10. Effect Of phosphorylation site mutants on microtubule-destabilizing activity.
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 vitro phosphorylated 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.
Antonsson
B
B
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 1228 Plan-les-Ouates, Switzerland.
Kassel
D B
DB
Di Paolo
G
G
Lutjens
R
R
Riederer
B M
BM
Grenningloh
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002352:Carrier Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins; D008868:Microtubule Proteins; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D008565:Membrane Proteins; D008868:Microtubule Proteins; D009414:Nerve Growth Factors; D010750:Phosphoproteins; C512848:Stmn2 protein, rat