Related Experiment Videos
L1 and N-CAM antibodies trigger protein phosphatase activity in growth cone-enriched membranes
S G Klinz1, M Schachner, P F Maness
1Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27599-7260, USA.
Abstract:
Triggering of the cell adhesion molecules L1 or N-CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N-CAM or specific antibodies decreases the steady-state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N-CAM in the growth cone-enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N-CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule-activated phosphatase. The most prominent substrates were a membrane-associated 200-kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N-CAM triggering. The antibody-induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N-CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N-CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N-CAM intracellular signaling pathways in growth cones.
Insights
Cell adhesion molecules L1 and N-CAM trigger phosphatases in nerve growth cones, dephosphorylating key proteins. This suggests protein phosphatases, not just kinases, are vital for L1 and N-CAM signaling.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Cell adhesion molecules (CAMs) like L1 and N-CAM are crucial for nerve growth cone function.
- Protein phosphorylation dynamics are critical for regulating growth cone behavior during neural development.
Purpose of the Study:
- To investigate the signaling pathways activated by L1 and N-CAM triggering in nerve growth cone membranes.
- To identify the specific enzymatic activities and protein substrates involved in L1 and N-CAM-mediated signaling.
Main Methods:
- Utilized a membrane fraction enriched in nerve growth cone components from fetal rat brain.
- Triggered L1 and N-CAM using purified proteins and specific antibodies targeting their extracellular regions.
- Analyzed changes in protein tyrosine and serine/threonine phosphorylation levels using biochemical assays.
- Tested the inhibitory effects of various phosphatase inhibitors on the observed dephosphorylation.
Main Results:
- Antibody-triggered activation of L1 and N-CAM elicited dephosphorylation of endogenous substrates.
- A 200-kDa membrane-associated protein and tubulin were identified as major substrates, dephosphorylated on tyrosine and serine/threonine residues.
- The antibody-induced phosphatase activity was sensitive to known tyrosine and serine/threonine phosphatase inhibitors.
- While some L1/N-CAM interactions inhibited tyrosine phosphorylation, others activated a phosphatase.
Conclusions:
- L1 and N-CAM triggering activates a phosphatase in growth cone membranes.
- Protein phosphatases play a significant role in intracellular signaling pathways downstream of L1 and N-CAM in growth cones.
- Both phosphatase activation and tyrosine kinase inhibition can occur upon L1 and N-CAM engagement.