Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A

Abstract

Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

“…Two of them resided in genes that were previously reported to be involved in paclitaxel resistance. One was a missense mutation located in RAPGEF4 that encodes a protein involved in microtubule polymerization and organization (33,34). The other was found in the 3′ UTR region of AMOTL1.…”

“…Three other RNA variants unique to the drug-tolerant single cells were found in genes that were involved in microtubule organization and stabilization during mitosis: RAPGEF4, NUDCD3, and KIAA1671 (Table 1). RAPGEF4 (Rap guanine nucleotide exchange factor 4) was previously shown to interact with protein complexes that were involved in microtubule polymerization and organization (33,34). RAPGEF4 protein is also known as exchange protein directly activated by cAMP 2 (EPAC2) and is one of the binding partners of MAP1A (microtubule-associated protein 1A) (33).…”

“…RAPGEF4 (Rap guanine nucleotide exchange factor 4) was previously shown to interact with protein complexes that were involved in microtubule polymerization and organization (33,34). RAPGEF4 protein is also known as exchange protein directly activated by cAMP 2 (EPAC2) and is one of the binding partners of MAP1A (microtubule-associated protein 1A) (33). MAP1A is known to promote elongation and nucleation of tubulin (35).…”

Microtubule-Associated Protein 1B-Light Chain 1 Enhances Activation of Rap1 by Exchange Protein Activated by Cyclic AMP but Not Intracellular Targeting

“…The MAP1A/LC2 gene is organized to encode a precursor polypeptide that undergoes proteolytic processing to generate the final 2556 amino acid MAP1A heavy chain and 249 amino acid LC2 polypeptide (Noiges et al, 2002). Using deletion analysis and GST-protein chimeras of individual EPAC1 domains, we have identified the CAMP domain of EPAC as being responsible for mediating interaction with LC2 (Magiera et al, 2004). Once bound to the CAMP domain, LC2 seems to increase the affinity of EPAC1 for cyclic AMP, thereby sensitizing catalytic activity toward Rap1 to lower concentrations of cyclic AMP (Gupta and Yarwood, 2005).…”

“…Using a combination of yeast two-hybrid analysis and coimmunoprecipitation, we have identified the light chain (LC) 2 of microtubule-associated protein (MAP) 1A as a novel protein-binding partner for EPAC1 and EPAC2 (Magiera et al, 2004). The MAP1A/LC2 gene is organized to encode a precursor polypeptide that undergoes proteolytic processing to generate the final 2556 amino acid MAP1A heavy chain and 249 amino acid LC2 polypeptide (Noiges et al, 2002).…”