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Phosphorylation of microtubule-associated protein-2 in GH3 cells. Regulation by cAMP and by calcium
1Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
Abstract:
The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.
Insights
Microtubule-associated protein-2 (MAP-2) in GH3 cells is phosphorylated by both cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase. These signaling pathways regulate MAP-2 phosphorylation at distinct sites.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- The GH3 rat pituitary cell line expresses a high molecular weight microtubule-associated protein (MAP-2).
- MAP-2 is characterized by its selective immunoprecipitation with MAP-2 antibodies and phosphorylation by specific kinases.
- MAP-2 is a minor cellular component but exhibits significant basal phosphorylation.
Purpose of the Study:
- To investigate the role of cAMP and Ca2+ signaling pathways in the phosphorylation of MAP-2 in GH3 cells.
- To identify the specific kinases involved in MAP-2 phosphorylation in response to cellular signaling.
- To elucidate the distinct phosphorylation sites regulated by different signaling pathways.
Main Methods:
- Immunoprecipitation of MAP-2 from GH3 cells.
- In vitro and in situ phosphorylation assays using cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.
- Phosphopeptide mapping to identify phosphorylation sites.
- Stimulation of GH3 cells with agents that modulate cAMP and Ca2+ levels.
Main Results:
- Vasoactive intestinal peptide, forskolin, IBMX, cholera toxin, and dibutyryl cAMP increased MAP-2 phosphorylation at specific sites without altering basal phosphorylation levels.
- Phosphopeptide mapping confirmed that cAMP kinase phosphorylates the same sites in vitro as those increased in situ upon cAMP pathway activation.
- Increased intracellular Ca2+ levels stimulated MAP-2 phosphorylation at distinct sites, which were identified by phosphopeptide mapping as those phosphorylated by CaM kinase in vitro.
Conclusions:
- Activation of cAMP- and Ca2+-dependent signaling pathways in GH3 cells leads to MAP-2 phosphorylation.
- cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase are the primary mediators of MAP-2 phosphorylation via these respective pathways.
- Distinct signaling pathways converge on specific phosphorylation sites of MAP-2, suggesting a complex regulatory mechanism.