MAP1A light chain 2 interacts with exchange protein activated by cyclic AMP 1 (EPAC1) to enhance Rap1 GTPase activity and cell adhesion.
We have recently demonstrated that light chain 2 (LC2) of the microtubule-associated protein MAP1A interacts with the cyclic AMP (cAMP)-binding domain of exchange protein directly activated by cyclic AMP 1 (EPAC1). In the present study we used a simultaneous expression system and found that LC2 enhances both basal and 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3':5'-cyclic monophosphate (8-CPT-2Me-cAMP)-stimulated Rap1 activation by EPAC1. LC2 is known to stabilize microtubules; therefore we examined whether microtubules enhanced Rap1 activation by LC2. Nocodazole inhibited Rap1 activity in cells transfected with EPAC1 alone but had little effect on Rap1 activity in cells transfected with both EPAC1 and LC2. This indicates that part of the actions of LC2 in enhancing EPAC1 activity may be through stabilization of microtubules. We also found that in cells transfected with LC2, Rap1 was more sensitive to activation by 8-CPT-2Me-cAMP. Moreover, LC2 enhanced the ability of transfected and endogenous EPAC1 to interact with cyclic AMP-agarose, indicating that LC2 elicits conformational changes in the cAMP domain of EPAC1, enhancing its ability to be activated by cyclic AMP. We also found that disruption of the interaction of endogenous EPAC1 and LC2 with antibodies to the cAMP domain of EPAC1 abolished Rap1 activity in PC12 cell lysates, demonstrating the importance of LC2 for EPAC1 activation in these cells. Consistent with a role of EPAC1 in controlling integrin activity, we found that cell adhesion to laminin was enhanced in LC2- and EPAC1-transfected cells stimulated with 8-CPT-2Me-cAMP. LC2 is therefore a biological enhancer of EPAC1 activity toward Rap1 and associated downstream signaling mechanisms.
Gupta
Mona
M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, United Kingdom.
Yarwood
Stephen J
SJ
D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005822:Genetic Vectors; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016023:Integrins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D016716:PC12 Cells; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D012685:Sepharose / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D016023:Integrins; C093429:MAP1A protein, human; D008869:Microtubule-Associated Proteins; C116122:RAPGEF3 protein, human; D012685:Sepharose; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D020827:rap1 GTP-Binding Proteins; D015739:Nocodazole