Essential role of the LIM domain in the formation of the PKCepsilon-ENH-N-type Ca2+ channel complex.
A LIM domain is a specialized double-zinc finger motif found in a variety of proteins. LIM domains are thought to function as molecular modules, mediating specific protein-protein interactions in cellular signaling. In a recent study, we have demonstrated that ENH, which has three consecutive LIM domains, acts as an adaptor protein for the formation of a functional PKCepsilon-ENH-N-type Ca2+ channel complex in neurons. Formation of this complex selectively recruits PKCepsilon to its specific substrate, N-type Ca2+ channels, and is critical for rapid and efficient potentiation of the Ca2+ channel activity by PKC in neurons. However, it is not clear whether changes in the local Ca2+ concentrations near the channel mouth may affect the formation of the triprotein complex. Furthermore, the molecular determinants for the interactions among these three proteins remain unknown. Biochemical studies were performed to address these questions. Within the physiological Ca2+ concentration range (0-300 microM), binding of ENH to the channel C-terminus was significantly increased by Ca2+, whereas increased Ca2+ levels led to dissociation of PKCepsilon from ENH. Mutagenesis studies revealed that the second LIM domain in ENH was primarily responsible for Ca2+-dependent binding of ENH to both the Ca2+ channel C-terminus and PKCepsilon. ENH existed as a dimer in vivo. PKCepsilon translocation inhibition peptide, which blocks the translocation of PKCepsilon from the cytosol to the membrane, inhibited the interaction between PKCepsilon and ENH. These results provide a molecular mechanism for how the PKCepsilon-ENH-N-type Ca2+ channel complex is formed and regulated, as well as potential drug targets to selectively disrupt the PKC signaling complex.
Chen
Yuan
Y
Department of Physiology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107, USA.
Lai
Meizan
M
Maeno-Hikichi
Yuka
Y
Zhang
Ji-fang
JF
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D010455:Peptides / Q000494:pharmacology; D051744:Protein Kinase C-epsilon / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats
D048868:Adaptor Proteins, Signal Transducing; D020864:Calcium Channels, N-Type; C102578:Pdlim5 protein, rat; D010455:Peptides; D051744:Protein Kinase C-epsilon; D002118:Calcium