A Dynamically Regulated 14–3–3, Slob, and Slowpoke Potassium Channel Complex in Drosophila Presynaptic Nerve Terminals

Abstract

Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

Alternative Splicing Switches Potassium Channel Sensitivity to Protein Phosphorylation

“…3, a and b). Importantly, the inhibitory effect of cAMP on STREX-S869A channels is dependent upon endogenous PKA activity as cAMP had no effect on the activity of STREX-S869A channels in the absence of ATP (n ϭ 5, not shown) or in the presence of PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] (n ϭ 6, Fig. 3b).…”

“…For example, in STREX-1 containing channels that are normally inhibited by PKA, dephosphorylation of the PKA site specifically within the STREX-1 insert would allow PKA-dependent activation of the channel through conserved sites in the C terminus (as for S4 STREX A construct). Further diversity and plasticity may be achieved through splice variant heteromultimerization and/or association with regulatory subunits (17)(18)(19).…”

“…Importantly, alternative splicing of the ␣-subunit is dynamically regulated in adults, for example during stress or pregnancy (15,16). Thus the diversity and plasticity of responses to PKA-dependent protein phosphorylation observed between BK channels in native tissues may result either from differential modulation of alternatively spliced BK channel ␣-subunits (12)(13)(14)(15) or through their interaction with different signaling complexes and ␤-subunits (17)(18)(19).…”

“…BK channels are regulated by multiple protein kinase signaling pathways (5,19,23,24). We have thus assayed the functional regulation of BK channel splice variants by directly activating PKA that remains closely associated with the channels in excised inside-out patches.…”

MONaKA, a Novel Modulator of the Plasma Membrane Na,K-ATPase

“…Protein in total cell lysates and streptavidin precipitates was detected by Western blotting with anti-HA and anti-Myc antibodies. Control cells were transfected with Myc-14-3-3, an intracellular protein that served as a negative control, as described previously (Zhou et al, 1999). The experiment was repeated three times.…”