title: Microtubule-associated protein 1B-light chain 1 enhances activation of Rap1 by exchange protein activated by cyclic AMP but not intracellular targeting.
abstract: We have previously demonstrated that EPAC1 interacts with light chain (LC) 2 of microtubule-associated protein (MAP) 1A. In the present study, we investigated whether the structurally related LC1 of MAP1B also interacts with EPAC1. We demonstrate that LC1 copurifies with EPAC1 from extracts of PC-12 cells, using cyclic AMP-agarose. Using recombinant LC1 and LC2 in pull-down and solid phase binding assays, we demonstrate direct interaction with a glutathione S-transferase-fusion of the cyclic AMP-binding (CAMP) domain of EPAC1. We also tested whether LC1 directed intracellular targeting of EPAC1 through its interaction with the CAMP domain. EPAC1 was found be in the soluble and particulate, nuclear/perinuclear fractions of cells. We found that the catalytic (CAT) domain of EPAC1, and not the CAMP domain, was responsible for recruitment to the nuclear/perinuclear fraction of cells. The targeting sequence responsible was located between amino acids 764 and 838 of EPAC1. Overexpresssion of an isolated CAT domain in COS1 cells was found to displace endogenous EPAC1 from the nuclear/perinuclear fraction, thereby inhibiting EPAC-activated Rap1 in this compartment. In contrast, LC1 was not able to compete for the binding of EPAC1 to this fraction. LC1, however, was able to enhance interaction of EPAC1 with cyclic AMP and heightened the ability of EPAC to activate Rap1. Antibody disruption of EPAC1/LC1 interaction in PC-12 cells ablated the ability of cyclic AMP to activate Rap1. LC1 is therefore not involved in intracellular targeting of EPAC1, but it is rather a molecular chaperone of EPAC activity toward Rap1.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Gillian
authors.initials: G
authors.lastname: Borland
authors.forename: Mona
authors.initials: M
authors.lastname: Gupta
authors.forename: Maria M
authors.initials: MM
authors.lastname: Magiera
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Rundell
authors.forename: Suzanne
authors.initials: S
authors.lastname: Fuld
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Yarwood
mesh_terms: D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D008869:Microtubule-Associated Proteins; C085284:microtubule-associated protein 1B; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins