Abstract
The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.
Highlights
The gap junction protein, connexin 43 (Cx43), plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication
Because 1) BMK1/ERK5, another mitogen-activated protein kinase (MAPK) family member activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit MEK1/ 2-ERK1/2 and MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF
We investigated the role of the activated MEK5-BMK1 module in Cx43 phosphorylation and subsequent inhibition of gap junctional communication (GJC)
Summary
Cx43, connexin 43; Cx43E, Cx43 with a C-terminal EGFP tag; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; BMK1, big MAP kinase 1; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; DsRed, Distal Red protein; GJC, gap junctional communication; FRAP, fluorescence recovery after photobleaching; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; DN, dominant negative; CA, constitutively active; EGF, epidermal growth factor; GST, glutathione S-transferase; MEF2C, myocyte elongation factor 2C; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; PD98059, MEK1 antagonist. Effectively inhibits MEK5, the upstream activator of BMK1 [7], sensitivity to this antagonist is insufficient to clearly define whether ERK1/2 or BMK1 mediates EGF-induced GJC uncoupling. We examined the specific role of BMK1 in EGF-induced phosphorylation of Cx43 and inhibition of GJC by using specific molecular reagents to manipulate the ERK1/2 or the BMK1 pathways independently in Cx43 heterologously expressed in human embryonic kidney (HEK) 293 cells. We found that BMK1 binds to Cx43 and phosphorylates preferentially on Ser-255 and a dominant negative form of BMK1 attenuates this phosphorylation in vivo These data demonstrate that BMK1 kinase activity alone is both a necessary and sufficient component in the mediation of EGF-induced Cx43 Ser-255 phosphorylation and subsequent inhibition of GJC
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
