slowpoke

Protein Interactions

Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, has been identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. It is possible that Slob serves to cluster dSlo and EAG together in a complex. Indeed, an interaction between EAG and Slowpoke is predicted by the finding that eag mutants in Drosophila exhibit altered calcium-dependent potassium currents at the larval neuromuscular junction (Schopperle, 1998).

Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. When Slob and dSlo are expressed together, there is a striking change in these subcellular distributions. Although some dSlo remains membrane associated, the two proteins colocalize in large doughnut-shaped structures that do not correspond obviously to known cellular organelles. The identity of these doughnut structures has not yet been determined, and it cannot be ruled out that they result from overexpression in a heterologous system. Time-lapse video microscopy shows that the structures are dynamic, moving within the cell and undergoing division into smaller structures. Similar structures have been described in cells coexpressing Shaker potassium channels and SAP97, a postsynaptic density protein. One intriguing possibility is that Slob is involved in channel protein processing, targeting, and/or degradation, and that the unidentified structures contain dSlo channels on their way to or from the plasma membrane. Slob may represent a new class of multi-functional channel-binding proteins (Schopperle, 1998).

To test the possibility that Slob might influence dSlo channel activity, patches were excised from cells expressing dSlo, and a preparation of purified glutathione-S-transferase-Slob (GST-Slob) fusion protein was applied to the cytoplasmic side of the patch. GST-Slob application strongly increases the steady-state open probability of dSlo channels. This effect is rapid in onset and reverses readily upon washing. GST-Slob also increases the peak dSlo current evoked by depolarizing voltage pulses. Because dSlo is a calcium-dependent channel, calcium was buffered carefully to insure that the activation by GST-Slob is not due to a change in calcium concentration. Furthermore, GST-Slob has no effect on the activity of hSlo, which is also calcium dependent. This lack of activation of hSlo is consistent with the finding that mSlo (which is virtually identical in amino acid sequence to hSlo) does not coimmunoprecipitate with Slob. These results support the conclusion that Slob can interact specifically with the dSlo channel to cause a change in its functional properties (Schopperle, 1998).

What is the in vivo function of Slob? Analysis of the protein sequence provides no obvious answers. The sequence of Slob has not been reported previously, but it does contain several well-characterized sequence motifs. For example, it contains a leucine zipper region that may allow it to form a complex with itself or other leucine zipper-containing proteins. Alternatively, Slob may be involved in signaling. The closest sequence matches to Slob are the signaling enzymes PKC and guanylate cyclase, but the matches are weak and the catalytic residues necessary for these enzymatic activities are not found in Slob. There is a PKC phosphorylation consensus site in the TRKQ cassette that can be removed by alternative splicing, suggesting possible regulation of Slob by phosphorylation. Another possibility is that Slob is an adaptor molecule. Finally, several proline-rich motifs that might bind SH3 domains including a Src kinase binding motif present in only the larger of the predicted amino-terminal splice variants, can be identified in the Slob sequence. It is possible that Slob is required as an adaptor to bring signaling proteins in close proximity to dSlo. Slob and Src have been found to coimmunoprecipitate from cotransfected cells. Taken together with the signaling protein binding domains within the Slob amino acid sequence, the effects on channel subcellular localization and gating properties suggest that Slob might link multiple channel properties to diverse upstream signals (Schopperle, 1998). COPY BELOW TO CAMKII

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 (Zhou, 1999).

The neuromuscular junction develops rapidly in Drosophila embryos (Broadie, 1993), and synaptic boutons and other morphological features of the synapse can be observed readily in mature larvae. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. A truncated Slob that lacks the amino-terminal 101 amino acids does not coimmunoprecipitate with 14-3-3. An examination of the amino acid sequence of Slob (reveals two motifs in this amino-terminal domain that resemble sequences in other 14-3-3-binding proteins. Although the downstream proline residue is lacking in the two Slob motifs, the participation of these motifs in 14-3-3 binding was examined by mutating the second serine in each motif (S54 and S79) to alanine. Both S54A Slob and S79A Slob bind less well to 14-3-3 than does wild-type Slob. When the two mutations are combined, no interaction of S54A/S79A Slob with 14-3-3 can be detected. The S54A/S79A Slob and wild-type Slob bind equally well to dSlo. Thus, the S54 and S79 residues in Slob are essential for its binding to 14-3-3 but not to dSlo (Zhou, 1999).

To test the hypothesis that the interaction of Slob and 14-3-3 might be regulated dynamically by phosphorylation in the fly, transgenic Drosophila that exhibit either higher or lower CaMKII activity than wild-type flies were used. The RQED1 fly line expresses a constitutively active rat CaMKII under the control of a heat shock promoter. In contrast, the ala2 fly line expresses a peptide inhibitor of CaMKII, also under heat shock control. Expression of constitutively active CaMKII increases the binding of Slob and 14-3-3 relative to the nonheat-shocked control. In contrast, expression of the peptide inhibitor of CaMKII decreases the binding compared with the nonheat-shocked control. These results demonstrate clearly that the interaction of Slob and 14-3-3 is not static but can be influenced rapidly by changes in CaMKII activity in the fly. Slowpoke channel activity was examined in detached membrane patches from transfected cultured cells to test the possibility that the channel might be modulated by 14-3-3. dSlo current evoked by a depolarizing voltage step to +30 mV is not affected by the coexpression of 14-3-3 and is somewhat larger when the channel is coexpressed with Slob. In contrast, much less dSlo current is evoked by the same depolarizing voltage step when all three proteins are expressed together, even though the GFP fluorescence confirms robust expression and membrane targeting of dSlo in these cells. The mean relative peak conductance (GRel) evoked by voltage steps to +30 mV was 0.64 ± 0.05 (mean ± SEM, n = 6) in patches from cells expressing dSlo alone, 0.70 ± 0.04 in patches from cells coexpressing dSlo and 14-3-3, 0.81 ± 0.03 in patches from cells expressing dSlo and Slob, but only 0.29 ± 0.09 in patches from cells expressing dSlo, Slob, and 14-3-3. Thus, the dSlo current evoked by depolarization to +30 mV is inhibited about 65% by 14-3-3. To determine whether 14-3-3 binding to Slob is required for this effect, dSlo and 14-3-3 were coexpressed with the S54A/S79A mutant Slob that can bind dSlo but does not bind to 14-3-3. dSlo current in patches from these cells is essentially identical to that found in patches from cells transfected with dSlo and Slob. Thus, the effect of 14-3-3 on dSlo current must be via Slob (Zhou, 1999).

To investigate the mechanism of this modulation, the voltage dependence of dSlo channel activity was examined. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity. This effect of 14-3-3 must be via Slob, because the voltage sensitivity in the presence of 14-3-3 and the S54A/S79A mutant Slob is identical to that seen when wild-type Slob is transfected alone. The shift in the voltage required for half-maximal activation (V1/2), elicited by 14-3-3, is 61 mV at 30 µM free calcium and is even larger at lower free calcium concentrations. These results also confirm the indication from the GFP fluorescence that dSlo channels are present in the membrane in the triply transfected cells, and it is their functional properties that are modulated by Slob and 14-3-3 (Zhou, 1999).

Large-conductance calcium-activated potassium channels (BK channels) are activated by depolarized membrane potential and elevated levels of intracellular calcium. BK channel activity underlies the fast afterhyperpolarization that follows an action potential and attenuates neurotransmitter and hormone secretion. Using a modified two-hybrid approach, the interaction trap, a novel protein from Drosophila, dSLIP1 (dSLo interacting protein), was identified which specifically interacts with Drosophila and human BK channels and has partial homology to the PDZ domain of alpha1 syntrophin. The dSLIP1 and dSlo mRNAs are expressed coincidently throughout the Drosophila nervous system, the two proteins interact in vitro, and they may be coimmunoprecipitated from transfected cells. Coexpression of dSLIP1 with dSlo or hSlo BK channels in Xenopus oocytes results in reduced currents as compared with expression of BK channels alone; current amplitudes may be rescued by coexpression with the channel domain that interacts with dSLIP1. Single-channel recordings and immunostaining of transfected tissue culture cells suggest that dSLIP1 selectively reduces Slo BK currents by reducing the number of BK channels in the plasma membrane (Xia, 1998b).

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. Protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch (Esguerra, 1994).

Drosophila Slowpoke (Slo) calcium-dependent potassium channels bind directly to the catalytic subunit of cAMP-dependent protein kinase (PKAc). Coexpression of PKAc with Slo in mammalian cells results in a dramatic decrease of Slo channel activity. This modulation requires catalytically active PKAc but is not mediated by phosphorylation of S942, the only PKA consensus site in the Slo C-terminal domain. Slo binds to free PKAc but not to the PKA holoenzyme that includes regulatory subunits and is inactive. Activators of endogenous PKA that stimulate Slo phosphorylation, but do not produce detectable PKAc binding to Slo, do not modulate channel function. Furthermore, the catalytically inactive PKAc mutant does bind to dSlo but does not modulate channel activity. These results are consistent with the hypothesis that both binding of active PKAc to dSlo and phosphorylation of dSlo or some other protein are necessary for channel modulation (Zhou, 2002).

The initial evidence on modulation of calcium-dependent potassium channels by closely associated protein kinases or phosphatases came from experiments in which K_Ca channels reconstituted from rat brain were activated in an artificial lipid bilayer by the addition of ATP. PKAc was identified as one of several protein kinases closely associated with Slo channels. To determine whether the bound PKAc modulates channel function, Slo currents were recorded in the whole-cell voltage-clamp configuration. There is a dramatic downregulation of Slo channel functional activity with cotransfection of PKAc. Although it has been reported that channel function is not affected by coexpression with both PKAc and Src, it is now known that Src enhances Slo channel activity, and this upregulation by Src may have masked the PKAc downregulation that is described in this study (Zhou, 2002).

The reduction of whole-cell Slo in PKAc-cotransfected cells represents a change in channel functional activity. Although it is difficult to obtain an accurate conductance-voltage relation because of the large current amplitude in the whole-cell configuration, both the lower current amplitude and slower activation kinetics of Slo currents in PKAc-cotransfected cells are consistent with a decreased sensitivity of the Slo channel to voltage and calcium. The changes in current do not appear to result from alterations in channel expression or membrane targeting, because the expression level of Slo protein, measured by quantitative Western blot, is actually several-fold higher, and its surface localization measured by immunocytochemistry is unchanged, in PKAc-cotransfected cells. In any event, it is difficult to explain the change in channel kinetics in terms of protein expression level (Zhou, 2002).

Although questions still remain about the precise molecular mechanism underlying the profound modulation of Slo channel activity by cotransfected PKAc, the studies suggest strongly that it requires the association between active PKAc and the Slo channel. However, Slo channel activity is not altered by cotransfection of a catalytically inactive PKAc (K72E), although this mutant PKAc is capable of binding to Slo. This demonstrates that the modulation of Slo activity is not simply the result of binding of PKAc per se, but also requires phosphorylation. This is also consistent with other reports of modulation of native and expressed Slo family channels by PKAc-dependent phosphorylation. However, results with forskolin suggest that phosphorylation, although necessary, is not by itself sufficient to produce modulation. Accordingly, the hypothesis that modulation requires phosphorylation, of either Slo or some other protein, by active PKAc bound to the channel, is favored (Zhou, 2002).

To determine the potential molecular target the phosphorylation of which might mediate the modulation of Slo activity, the role of serine 942 in the Slo C-terminal domain was examined. This residue has long been recognized as a consensus PKA substrate, and it is readily phosphorylated by PKA both in vitro and in vivo. Interestingly, it was found that the mutation of serine 942 to alanine does not affect Slo binding to or modulation by PKAc. This is consistent with a previous report showing that S942A Slo is not different from the wild-type channel in its kinetic variability when expressed in Xenopus oocytes. Although it is convenient to use the anti-pS942 antibody to measure channel phosphorylation, this is not meant to imply that S942 participates in Slo modulation by PKAc (Zhou, 2002).

In addition to S942, other serine and threonine residues are thought to be exposed to the intracellular milieu. To identify other PKA substrate sites on Slo, an in vitro phosphorylation assay was performed using a recombinant fusion protein containing the entire Slo C-terminal domain. This domain is considerably longer than many other voltage-dependent potassium channels and constitutes approximately two-thirds of the total Slo channel protein. Somewhat surprisingly, it was found that other than S942, no additional amino acid in the entire Slo C-terminal domain is phosphorylated by PKAc in vitro. However, PKA phosphorylation of several serine or threonine residues in the intracellular loops between transmembrane domains has not been examined. It is equally plausible that PKAc phosphorylates other proteins that may themselves or through some signaling pathway modulate Slo channel activity. Slo is indeed regulated by several closely associated proteins. At least one Slo-interacting protein, 14-3-3, interacts with Slo via the adaptor protein Slob in a phosphorylation-dependent manner, and formation of this protein complex results in inhibition of channel activity. A recent report also showed that a Slo channel associating protein, cPLA2-alpha, is a target for phosphorylation. Phosphorylation of cPLA2-alpha results in activation of the channel, whereas phosphorylation of other regulatory elements causes channel inactivation. Finally, Slo channel activity is not modulated by activators of endogenous PKA, including forskolin and cBIMPS, which do not enhance binding of PKAc to Slo. Although these results do not lend themselves readily to unequivocal interpretation, they are consistent with the hypothesis that whatever the actual substrates of PKA are, targeting of sufficient PKAc via binding to Slo is also a requirement for channel modulation (Zhou, 2002).

It has been well documented that PKA forms regulatory complexes with some ion channels and ligand-gated receptors. PKA targeting is often achieved through AKAPs, proteins that bind to the PKA regulatory subunit as well as the substrate. However, overlay experiments showed a direct binding between Slo and PKAc, suggesting a novel channel-PKA protein complex. The present studies support this hypothesis and provide additional molecular details about this regulatory complex. Using co-immunoprecipitation approaches, it was shown that Slo binds only to free PKAc but not to the PKA holoenzyme, and that both PKA regulatory subunit and PKI inhibit the association between Slo and PKAc. A 35 amino acid region has been identified in the Slo C-terminal domain that is essential for PKAc binding. Interestingly, this region includes the consensus PKA substrate site, S942, although residues within the substrate site itself (RRXS) do not appear to be critical for binding to PKAc. This demonstrates that the Slo-PKAc association is not simply an enzyme-substrate complex. It is also consistent with previous studies on interactions between PKAc and regulatory subunits or PKI that show that residues separate from the pseudosubstrate site of the regulatory subunits or PKI are important in mediating the high-affinity binding to PKAc. Moreover, in vitro phosphorylation results suggest that binding of Slo to PKAc does not prevent the enzyme from phosphorylating its substrates. It remains to be determined how the activity of Slo-bound PKAc is regulated in cells (Zhou, 2002).

It is noteworthy that channels of the Slo family can physically associate with other protein kinases, including the Src tyrosine kinase and type I cGMP-dependent protein kinase. This suggests that modulation of channel activity may involve multiple regulatory mechanisms. Further biochemical and electrophysiological studies to dissect these regulatory pathways will undoubtedly provide important insights into the relationship between various signal transduction pathways and neuronal physiology (Zhou, 2002).

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