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Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase,
N Sahyoun1, O B McDonald, F Farrell
1Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Abstract:
A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.
Insights
CaM kinase Gr selectively phosphorylates Rap-1b, a brain protein, linking neuronal signaling to GTP-binding proteins. This phosphorylation is reversible, suggesting roles in neuronal functions like neurotransmitter release.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Signaling
Background:
- Low molecular weight GTP-binding proteins, such as Rap-1b, are crucial in neuronal function.
- Neuronal signaling pathways involving Ca2+ play vital roles in brain activity.
Purpose of the Study:
- To investigate the specific interactions between CaM kinase Gr and Rap-1b.
- To elucidate the role of CaM kinase Gr in the phosphorylation of Rap-1b and its potential implications in neuronal signaling.
Main Methods:
- In vitro phosphorylation assays using purified CaM kinase Gr and Rap-1b.
- Characterization of the phosphorylation site on Rap-1b.
- Investigation of the effect of CaM kinase Gr autophosphorylation on Rap-1b phosphorylation rate.
- Testing the specificity of CaM kinase Gr towards other Ras superfamily proteins.
- Assessing the reversibility of Rap-1b phosphorylation using a brain phosphoprotein phosphatase.
Main Results:
- CaM kinase Gr selectively phosphorylates Rap-1b at a serine residue near its carboxyl terminus with a stoichiometry of approximately 1.
- Both CaM kinase Gr and cAMP-dependent protein kinase phosphorylate Rap-1b, but CaM kinase II does not.
- Autophosphorylation enhances the rate of Rap-1b phosphorylation by CaM kinase Gr.
- CaM kinase Gr does not phosphorylate other tested Ras superfamily proteins (Rab-3A, Rap-2b, c-Ha-ras p21).
- Phosphorylation of Rap-1b is reversible by an endogenous brain phosphoprotein phosphatase.
Conclusions:
- CaM kinase Gr is a key enzyme in the phosphorylation of Rap-1b, a brain-enriched GTP-binding protein.
- This phosphorylation event establishes a link between neuronal Ca2+ signaling pathways and Rap-1b.
- The findings suggest that Rap-1b phosphorylation by CaM kinase Gr may regulate critical neuronal processes such as transmembrane signaling, vesicle transport, and neurotransmitter release.