Monomeric 14-3-3 Protein Is Sufficient to Modulate the Activity of the Drosophila Slowpoke Calcium-dependent Potassium Channel

Yi Zhou,Smitha Reddy,Heather Murrey,Hong Fei,Irwin B Levitan

doi:10.1074/jbc.m211907200

Yi Zhou, Smitha Reddy + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m211907200

Copy DOI

Abstract

Drosophila 14-3-3zeta (D14-3-3zeta) modulates the activity of the Slowpoke calcium-dependent potassium channel (dSlo) by interacting with the dSlo binding protein, Slob. We show here that D14-3-3zeta forms dimers in vitro. Site-directed mutations in its putative dimerization interface result in a dimerization-deficient form of D14-3-3zeta. Both the wild-type and dimerization-deficient forms of D14-3-3zeta bind to Slob with similar affinity and form complexes with dSlo. When dSlo and Slob are expressed in mammalian cells, the dSlo channel activity is similarly modulated by co-expression of either the wild-type or the dimerization-deficient form of D14-3-3zeta. In addition, dSlo is still modulated by wild-type D14-3-3zeta in the presence of a 14-3-3 mutant, which does not itself bind to Slob but forms heterodimers with the wild-type 14-3-3. These data, taken together, suggest that monomeric D14-3-3zeta is capable of modulating dSlo channel activity in this regulatory complex.

Highlights

14-3-3 proteins comprise a ubiquitous family of highly conserved proteins
We have shown that D14-3-3␨ forms a regulatory protein complex with the Drosophila calcium-dependent potassium channel,1 mediated by its binding to a novel channel binding protein, Slob [13, 14]
We show that modulation of dSlo channel activity by wild-type D14-3-3␨ is not affected by overexpression of another 14-3-3␨ mutant, which can heterodimerize with wild-type 143-3 but does not itself bind to Slob

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Rabbit polyclonal anti-dSlo, anti-Slob, and anti-14-3-3 antibodies were generated and purified as described previously [13, 14]. Monoclonal anti-FLAG antibody (M2) was purchased from Sigma. Anti1D4 monoclonal antibody was kindly provided by Dr D. CDNA Constructs and Mutagenesis—The 14-3-3 dimerization-deficient mutants were created using site-directed mutagenesis of the following D14-3-3␨ amino acids: L15AE to Q15QR and R88VE to N88VQ. A PCR strategy with appropriate primers was used to introduce the pertinent mutations and to generate 1D4 or FLAG epitope tags on the C terminus of D14-3-3␨ cDNA. The PCR products were cloned into the mammalian expression vector, pcDNA3. Wild-type and dimerization-deficient mutant 14-3-3␨-EBFP cDNAs were constructed by cloning

14-3-3 Monomer Modulates dSlo Channel Activity

RESULTS

DISCUSSION