Phosphorylation-dependent Binding of 14-3-3 Proteins Controls TRESK Regulation

Abstract

The two-pore domain K ؉ channel, TRESK (TWIK-related spinal cord K ؉ channel) is reversibly activated by the calcium/ calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3 with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3 accelerated the return of the K ؉ current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3 was also effective, when it was tethered to the channel by a flexible polyglutaminecontaining chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The ␥ and isoforms of 14-3-3 controlled TRESK regulation, whereas the ␤, , ⑀, , and isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K ؉ channel family.Members of the 14-3-3 family are dimeric proteins, and each subunit possesses a single polypeptide binding groove (1). Proteins that bind these grooves typically encode either the RSXpSXP (mode I) or RX(Y/F)XpSXP (mode II) consensus motif (where X may be any amino acid, and pS denotes phosphoserine (2, 3). Several different interacting partners of 14-3-3 have been described, and 14-3-3 proved to be an important constituent of large protein complexes implicated in such diverse processes as the initiation of DNA replication, transcription, control of cell cycle, intracellular trafficking, and the modulation of ion channel function (4, 5).Two-pore domain potassium (2PK ϩ ) channels give rise to background (leak) K ϩ currents that are pivotal regulators of the excitability in neurons and other cell types (6). Members of this potassium channel family attracted particular attention as the stimulation of their currents essentially contributed to the therapeutically important action of volatile anesthetics (7-10). Among the 15 2PK ϩ channels, so far only TASK-1 and TASK-3 subunits have been shown to interact with 14-3-3␤ andthrough an unconventional (mode III) C-terminal motif. The binding of 14-3-3 overrides the endoplasmic reticulum retention signal and redirects these TASK channels to the cell surface (11-13).TRESK, 2 the 15th member of the 2PK ϩ channel family, was cloned from human spinal cord (14) and mouse cerebellum (15). Its mRNA is also expressed in the testis (15), spleen, thymus, placenta (16), and in the cerebrum (9). Recently, TRESK ...

TRESK Background K+ Channel Is Inhibited by Phosphorylation via Two Distinct Pathways

“…14-3-3 binds to the RSNSCPE motif (a typical mode 1 site) in the intracellular loop of the channel, if the second (underlined) serine (Ser-264 in mouse TRESK) is phosphorylated (21). The interaction between TRESK and 14-3-3 has a functional consequence dif-ferent from that of the previously described association of TASK-1 or TASK-3 K2P channels with the adaptor protein.…”

“…After the calcineurin-dependent activation, return of TRESK current to the resting state is impeded by 14-3-3 (21). Originally, we proposed that the binding of 14-3-3 to the phosphorylated Ser-264 obstructed the rephosphorylation of Ser-276 after its calcineurin-mediated dephosphorylation, and thus 14-3-3 interfered with the recovery (21). However, analysis of the effect of 14-3-3 on TRESK channels mutated at distinct regulatory serine residues indicated a more complex mechanism of action of the adaptor protein.…”

“…the expression of the channels is enhanced (22)(23)(24)(25). According to a recent study, N-glycosylation modulates the surface expression of TRESK (26), and we have shown that 14-3-3 regulates TRESK channel activity (21). After the calcineurin-dependent activation, return of TRESK current to the resting state is impeded by 14-3-3 (21).…”

Protein Kinase A Is Central for Forward Transport of Two-pore Domain Potassium Channels K2P3.1 and K2P9.1

“…14-3-3 has also been shown to play a role in the intracellular transport of a number of other membrane proteins, including the K ATP channel Kir6.2, invariant chain protein, nicotinic acetylcholine receptor (nAChR␣4), kainate receptor KA2, and G-protein-coupled receptor 15 (11,26). Furthermore, another K 2P channel member (K 2P 18.1/TRESK) has recently been shown to be negatively regulated by 14-3-3 in a phosphorylation-dependent manner (27).…”

The LQLP Calcineurin Docking Site Is a Major Determinant of the Calcium-dependent Activation of Human TRESK Background K+ Channel

“…These residues are also conserved in human TRESK as Ser-252, Ser-262, and Ser-264. The first serine is phosphorylated by protein kinase A (10), and this reaction permits the binding of 14-3-3 adapter protein (10,11). Microtubule-affinity regulating (MARK) kinases are currently the only known enzymes that phosphorylate the other two serines of functional importance.…”