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Ectopic Potassium Uptake in trk1 trk2 Mutants ofSaccharomyces cerevisiae Correlates with a Highly Hyperpolarized Membrane Potential*

Null trk1 trk2 mutants ofSaccharomyces cerevisiae exhibit a low-affinity uptake of K+ and Rb+. We show that this low-affinity Rb+ uptake is mediated by several independent transporters, and that trk1Δ cells and especially trk1Δ trk2Δcells are highly hyperpolarized. Differences in the membrane potentials were assessed for sensitivity to hygromycin B and by flow cytometric analyses of cellular DiOC6(3) fluorescence. On the basis of the latter analyses, it is proposed that Trk1p and Trk2p are involved in the control of the membrane potential, preventing excessive hyperpolarizations. K+ starvation and nitrogen starvation hyperpolarize both TRK1 TRK2 and trk1Δ trk2Δ cells, thus suggesting that other proteins, in addition to Trk1p and Trk2p, participate in the control of the membrane potential. The HAK1 K+ transporter fromSchwanniomyces occidentalis suppresses the K+-defective transport of trk1Δ trk2Δ cells but not the high hyperpolarization, and the HKT1 K+transporter from wheat suppresses both defects, in the presence of Na+. We discuss the mechanism involved in the control of the membrane potential by Trk1p and Trk2p and the causal relationship between the high membrane potential (negative inside) of trk1Δ trk2Δ cells and its ectopic transport of alkali cations.

Potassium is an indispensable element, which is accumulated against large transmembrane concentration gradients in cells living in diluted environments. Probably because of the central role of this element in all forms of life, different mechanisms mediating K ϩ uptake have evolved in different types of cells. In plants and fungi, the cellular uptake of K ϩ is probably always an electrophoretic process, which takes place in response to the membrane potential created by the H ϩ -pump ATPase (1, 2), as described for Neurospora crassa (3).
Among all eucaryotic non-animal cells, the maximum information about potassium transport has been obtained in Saccharomyces cerevisiae. In this fungus, the biochemistry (4 -7) and the genetics of K ϩ transport (8 -11) and H ϩ -pump ATPase (12) have been extensively studied. The TRK1 gene of S. cerevisiae encodes a notable K ϩ transporter, which is adapted to provide the required amount of K ϩ in many different nutritional conditions. To perform this function, this transporter changes its K m , which shows values in the millimolar range in cells growing at millimolar concentrations of K ϩ , and as low as 20 -30 M in K ϩ -starved cells (5,8,13). The V max is also variable, depending on the cellular pH and K ϩ content of the cells (5,6). A second gene, TRK2 (10,11), encodes a second K ϩ transporter structurally related to Trk1p (11). However, unlike Trk1p, Trk2p shows a very low Rb ϩ influx V max in trk1⌬ cells with a low K ϩ content, and the influx is undetectable in TRK1 cells or in trk1⌬ cells with a normal K ϩ content (7). Using a trk1⌬ strain overexpressing Trk2p, it has been found that the affinity of the TRK2 transporter for K ϩ and Rb ϩ is also regulated by the K ϩ content of the cell, and that the K m in K ϩstarved cells is only slightly higher than that of TRK1 (7). Because the V max of TRK2 is very low in comparison to that of TRK1 and the K m is higher than the K m of TRK1, the function of Trk2p seems to be superfluous as an independent K ϩ transporter.
Deletion of the TRK1 and TRK2 genes results in cells that grow normally at high concentrations of K ϩ , and slowly at relatively low concentrations, exhibiting a Rb ϩ influx kinetics with a K m of 60 mM and a V max of 9 and 16 nmol mg Ϫ1 min Ϫ1 , in the two strains studied so far (7). This low-affinity Rb ϩ uptake of trk1⌬ trk2⌬ cells also constitutes the major pathway for Rb ϩ uptake in trk1⌬ TRK2 cells, in which Trk2p mediates a minor pathway, as already mentioned. However, in contrast with the relevance of the low-affinity K ϩ uptake in trk1⌬ TRK2 and trk1⌬ trk2⌬ cells, this uptake has never been detected in wild strains (4,5). Because the K m of the low-affinity Rb ϩ influx is much higher than the K m of TRK1 and the V max not insignificant in comparison to the V max of TRK1, it can be concluded that the low-affinity Rb ϩ uptake has not been detected in TRK1 cells because it does not exist in these cells.
Therefore, two important questions remain unanswered about K ϩ transport in S. cerevisiae, the function of Trk2p and the identity, and consequently the function, of the transporter mediating the low-affinity uptake of K ϩ and Rb ϩ observed in trk1⌬ cells. For the latter question two hypotheses have been put forward, that this transport takes place mediated by several non-K ϩ transporters, which harbor intrinsic K ϩ transport capabilities (14 -16), and that it is mediated by a part of the normal K ϩ uptake system, which is multimeric and loses its normal properties in the absence of Trk1p (7). The former hypothesis is supported by well documented studies with strains carrying mutations in sugar and amino acid transporters, and with strains overexpressing amino acid permeases (14 -16). The latter has been advanced to explain why the low-affinity Rb ϩ uptake does not exist in wild strains, and to explain why glucose activates the low-affinity Rb ϩ influx of trk1⌬ mutants in the same fashion as it does in wild strains (7,17).
In this paper we report the characteristics of Rb ϩ uptake, tolerance to hygromycin B, and cellular DiOC 6 (3) fluorescence, as determined by flow cytometry, in strains carrying different combinations of wild and null alleles of the TRK1 and TRK2 genes. The results suggest that Trk1p and Trk2p are involved in the regulation of the electrical membrane potential. Deletion of these proteins brings the cells to a very high membrane potential, negative inside, which could drive an ectopic uptake of alkali cations.

EXPERIMENTAL PROCEDURES
Strains, Plasmids, Growth Conditions, and Methods-The S. cerevisiae strains used in this study are all isogenic to W303.1A (Mata ura3 his3 leu2 ade2 trp1) (Table I).
Cells were grown in complex YPD or YPG (1% yeast extract, 2% peptone, 2% glucose or galactose) media, and in mineral media containing either ammonium or arginine as nitrogen sources, as described previously (5). The mineral media contained 5 M K ϩ and 200 M Na ϩ , and KCl and NaCl were added in each case to obtain the required amounts of K ϩ and Na ϩ . Cells with a normal K ϩ content were routinely grown in the arginine medium supplemented with 0.5 mM K ϩ for the TRK1 TRK2 and TRK1trk2⌬ strains, 2.5 mM K ϩ for the trk1⌬ TRK2 strain, and 10 mM K ϩ for the trk1⌬ trk2⌬ strain. K ϩ -starved cells were obtained by suspending cells with a normal K ϩ content in K ϩ -free arginine medium for 3 h (5). The mineral media for nitrogen or phosphate starvation were prepared substituting K ϩ for arginine or sulfuric acid for phosphoric acid, respectively; for nitrogen starvation, amino acid supplementation, if required, was reduced 10-fold. Starvation was achieved by incubating the cells for 5 h in these media. trk1⌬ trk2⌬-pGALHAK1 cells were grown in YPG and transferred to YPD as indicated in each case. Hygromycin B resistance was tested in YPD.
N. crassa was grown as described elsewhere (23). Cells with a normal K ϩ content were grown at 3 mM K ϩ . To obtain low-K ϩ cells, the conidia were grown at 50 M K ϩ for 6 h and then transferred to 10 M K ϩ for 1 h. All procedures for manipulating DNA were standard ones (24).
Rb ϩ Uptake-Cells were separated from the culture medium by centrifugation, washed, and suspended in testing buffer composed of 10 mM MES 1 buffer brought to pH 6.0 with Ca(OH) 2 , containing 0.1 mM MgCl 2 and 2% glucose. In some experiments with trk1⌬ trk2⌬-pR-SHAK1 strain carrying the pGAL1-HAK1 gene, galactose-substituted glucose, and in the experiments designed to test the effect of divalent cations on Rb ϩ influx, the pH of the testing buffer was adjusted with NaOH, and MgCl 2 was withdrawn. To start the experiments, Rb ϩ was added to the cell suspension and, at short intervals, samples of cells were removed by filtration from the testing buffer. To determine the Rb ϩ content, the cells were treated with HCl and the extracts analyzed by atomic emission spectrophotometry, as described previously (5). In all cases, we determined the initial rate of Rb ϩ uptake from the time course of the net accumulation.
Flow Cytometry-S. cerevisiae and N. crassa cells were grown as described in each case. S. cerevisiae was always grown at low cell density (Ͻ0.3 mg dry weight ml Ϫ1 ), and as a consequence the concentration of glucose was very similar in all experiments (close to 2%). Cells were harvested from the culture medium, suspended in testing buffer (2 ϫ 10 6 cells ml Ϫ1 ), and exposed to DiOC 6 (3) cyanine dye (3,3Ј-dihexyloxacarbocynine iodide, Molecular Probes, Eugene, OR) at 1 nM for 30 min at 28°C, in the dark. To test yeast cell viability, propidium iodide was added at the moment of the analysis. Flow cytometric analyses were performed in a FACScan (Becton Dickinson) and EPICS XL (Coulter Electronics) flow cytometers, equipped with argon lasers. For the determination of the DiOC 6 (3) fluorescence, excitation at 488 nm and a 525-nm dichroic LP filter were used. For propidium iodide, a 590-nm dichroic filter and a 610-nm LP absorbance filter were added. Because the cellular fluorescence was size dependent, we normalized all the results measuring the fluorescence of single small cells (25). In all experiments, a control sample of wild-type cells (W303.1A strain) grown in the arginine medium at 0.5 mM K ϩ was analyzed in parallel with the other samples, and the fluorescence values given by the flow cytometer were always referred to the fluorescence of the control cells and expressed as a percentage. All measurements were repeated at least in three different days.

RESULTS
Characteristics of the Low-affinity K ϩ Transport of trk1⌬ trk2⌬ Cells-The kinetic study of the influx of the alkali cations in trk1⌬ trk2⌬ cells with different K ϩ contents and the competitive inhibitions exerted between them indicated that these cells took up alkali cations with little differences in affinities (we determined K m values of 60 mM K ϩ , 60 mM Rb ϩ , 110 mM Na ϩ , and 100 mM Li ϩ in the W⌬3 strain), and without regulation by the K ϩ content. Although this uptake shows homogeneous kinetics, experiments with different inhibitors showed that it involves different pathways. Ammonium inhibited 45% of the low-affinity Rb ϩ uptake of trk1⌬ trk2⌬ cells, but showed YEp352 derivative containing the HXT3 gene This study no effect over the remaining 55%; low concentrations of Mn 2ϩ , and to a lesser extent other divalent cations (Mg 2ϩ , Ca 2ϩ , and Ba 2ϩ ), inhibited 30%, but showed no effect on the remaining 70% ( Fig. 1); and ammonium plus Mn 2ϩ (20 mM each) inhibited 60% of the uptake. These results suggest the existence of at least three pathways for Rb ϩ uptake in trk1⌬ trk2⌬ cells, one inhibited by Mn 2ϩ , another inhibited by ammonium and insensitive to Mn 2ϩ , and a pathway insensitive to both ions. It is worth mentioning that the described inhibition of Rb ϩ uptake by ammonium or Mn 2ϩ in trk1⌬ trk2⌬ cells was different from that found in wild-type cells. In the latter, ammonium was a competitive inhibitor of Rb ϩ uptake, and Mn 2ϩ and other divalent cations did not show any significant effect (not shown). Rb ϩ uptake in trk1⌬ trk2⌬ cells was also found to be very sensitive to uncouplers, much more than in TRK1 TRK2 or TRK1 trk2⌬ cells. These experiments were carried out in K ϩstarved cells, in order to test cells in identical physiological conditions, finding that at 20 M CCCP, Rb ϩ influx (initial rate of uptake at 100 mM Rb ϩ ) in trk1⌬ trk2⌬ cells was inhibited by 80 -100%, whereas this concentration showed no effect on TRK1 TRK2 cells (Fig. 2).
Overexpression of Hxt3p and Hnm1p Partially Suppresses the Phenotype of trk1⌬ trk2⌬ Cells-In a different part of the study of the low-affinity K ϩ uptake of S. cerevisiae, we had isolated mutants suppressing the trk1⌬ trk2⌬ phenotype, and found that many of them mapped on the HXT3 gene, as is consistent with previous reports (14,15,26). The sequence of the HXT3 mutated genes revealed that some of these mutations were located in the 5Ј non-coding region. This suggested that these mutations produced overexpression of the encoded protein, and hence that the wild HXT3 transporter was able to transport K ϩ . To test this possibility, we inserted the HXT3 gene in a multicopy plasmid and transformed it into the trk1⌬ trk2⌬ strain, finding that the growth at low K ϩ was improved significantly (not shown).
Considering this result, we searched for other genes which, when overexpressed, suppress the trk1⌬ trk2⌬ phenotype. For this purpose, we transformed a trk1⌬ trk2⌬ strain with a library of genomic DNA in a multicopy plasmid and screened for clones suppressing the high K ϩ requirement of trk1⌬ trk2⌬ cells. Using the ammonium medium with 2 mM K ϩ , we isolated a clone with a 9-kilobase insert containing the YGL077c open reading frame, which encodes the HNM1 choline transporter (27). By subcloning this gene alone in a multicopy plasmid, we found that HNM1 partially suppressed the trk1⌬ trk2⌬ pheno-type (not shown). Although the overexpression of Hxt3p or Hnm1p improved the growth rate of the mutant, it increased less than 10% the rate of Rb ϩ uptake, suggesting that these proteins made a small contribution to the total K ϩ uptake of trk1⌬ trk2⌬ cells.
The finding that the overexpression of the HXT3 and HNM1 transporters partially suppressed the trk1⌬ trk2⌬ phenotype, as it is the case of some amino acids and the inositol transporters (16), gave clear support to the notion that multiple K ϩ pathways, possibly making small individual contributions, composed the low-affinity K ϩ uptake in trk1⌬ and trk1⌬ trk2⌬ mutants. Because the low-affinity K ϩ uptake does not exist in wild strains, the question was how the lack of one protein, Trk1p, could open all these pathways.
The Lack of Trk1p Triggers the Low-affinity Uptake of Rb ϩ -Two different reasons could trigger the general response that furnished several different transporters with the capacity of transporting K ϩ , the loss of Trk1p by itself or the K ϩ deficiency generated by its loss. To distinguish between these two possibilities, we used the trk1⌬ trk2⌬ strain transformed with the HAK1 gene of Schwanniomyces occidentalis (28). This gene encodes a K ϩ transporter not related to Trk1p and Trk2p, that restores K ϩ uptake, and consequently a normal growth at low K ϩ , in trk1⌬ trk2⌬ cells (28). Therefore, if trk1⌬ trk2⌬ cells expressing Hak1p still kept the low-affinity K ϩ and Rb ϩ uptake, the mediation of the K ϩ deficiency in this uptake could be ruled out.
For a high expression of Hak1p in S. cerevisiae, we used two different constructs, placing the coding region of the HAK1 gene under the control of the PGK1 or GAL1 gene promoters. Both constructs transformed into the trk1⌬ trk2⌬ strain allowed the transformants to grow at K ϩ concentrations even lower than those needed for TRK1 strains. In contrast, in these experiments the low-affinity uptake was present or absent depending on the testing conditions. In the pPGK1-HAK1 transformant, Rb ϩ influx in K ϩ -starved cells showed only one kinetic component, corresponding to HAK1, whereas in normal K ϩ cells, Rb ϩ influx showed both the HAK1 and the lowaffinity kinetic components. In the pGAL1-HAK1 transformant, cells grown on galactose (YPG) and tested on galactose showed only the Rb ϩ -influx kinetics of HAK1, but shortly after the transfer to glucose (YPD) they showed both the HAK1 and the low-affinity kinetic components. A longer incubation in glucose progressively decreased the former without affecting the latter (not shown). trk1⌬ trk2⌬ Strains Are Hypersensitive to Hygromycin B-The results obtained with the HAK1 transporter indicated that Hak1p performed the K ϩ transport functions of Trk1p, but that only in very special conditions did it prevent the lowaffinity Rb ϩ uptake. Among all the possible causes for this behavior, changes in the membrane potential were the most likely. The working hypothesis was that trk1⌬ trk2⌬ cells could be hyperpolarized, and that the low-affinity Rb ϩ uptake required such hyperpolarization. This hypothesis gave a satisfactory explanation to the high sensitivity of the low-affinity Rb ϩ uptake to uncouplers (Fig. 2), and was testable by determining the resistance of the different strains to hygromycin B, an amino glycoside antibiotic for which, as in the case of Dio-9 (29,30), the resistance of the cells depends on their membrane potential (31,32). The tests were performed in YPD, which contains sufficient K ϩ (15 mM) for normal growth of the trk1⌬ trk2⌬ strain, using the different mutants in the TRK1 and TRK2 genes, and the trk1⌬ trk2⌬ strain transformed with the pPGK1-HAK1 gene or the HKT1 cDNA under the control of the PMA1 promoter (Table II). HKT1 encodes a K ϩ -Na ϩ symporter in wheat (33)(34)(35), which is related to the TRK transporters (36). Transformation with HKT1 suppressed the K ϩ uptake deficiency of our trk1⌬ trk2⌬ strain, consistent with previous results (33)(34)(35). The tests of hygromycin B resistance (Table II) showed that the trk1⌬ strains, either TRK2 or trk2⌬, were inhibited by one-fifth of the concentration tolerated by the TRK1 strains, and that HKT1 but not HAK1 suppressed the hypersensitivity of the trk1⌬ trk2⌬ strain. In pma1 mutants the sensitivity to hygromycin B depends on the K ϩ content of the medium (31), and we found the same effect in trk mutants. However, even after the addition of 50 mM K ϩ to the YPD testing medium, the differences were still appreciable (Table  II).
The different sensitivity to hygromycin B of the trk1⌬ and TRK1 cells in a medium which does not impose a K ϩ limitation on trk1⌬ strains indicated that hypersensitivity to hygromycin B could not be a direct consequence of K ϩ deficiency. The same conclusion could be reached considering that trk1⌬ trk2⌬ cells expressing Hak1p were also sensitive. Although these cells can grow at K ϩ concentrations one thousand times lower than those existing in YPD (28) they were sensitive to hygromycin B. Taken together, these results suggested that the trk1⌬ mutation was associated with a hyperpolarized plasma membrane, and that this defect could be suppressed by the expression of some K ϩ transporters but not by all of those able to complement the K ϩ uptake deficiency.
Differences in the Membrane Potential Can Be Estimated by DiOC 6 (3) Fluorescence and Flow Cytometry-The electrical membrane potential of yeast cells is not known, but comparative assessments of membrane potentials in different conditions may be obtained with fluorescent cyanine dyes (37,38). The cyanine dye DiOC 6 (3) has been used to stain internal membranes in yeast (39) and to detect dysfunctional mitochondria (18,25,40). To use this dye as a probe for the membrane potential, avoiding the interference of the fluorescence of the internal membranes, we reduced the concentration of the dye in the staining medium (testing buffer) to 1 nM and tested only fermenting cells in which the function of the mitochondria is highly reduced (41). Fermenting cells stained with 1 nM DiOC 6 (3) and analyzed by flow cytometry showed size-dependent fluorescence (Fig. 3A). Taking the whole population or dividing it into two subpopulations, roughly corresponding to single cells and cells with small buds, and large cells and cells with large buds, K ϩ -starved cells showed a much higher fluorescence than cells with a normal K ϩ content (Fig. 3, A and B).
To investigate the contribution of the mitochondria to the overall fluorescence of the cells, we performed some preliminary experiments with rho ϩ and rho o strains, finding that in cells growing at 2% glucose and stained with 1 nM DiOC 6 (3) the difference in fluorescence between the rho ϩ and rho o strains was negligible. Then we used a yeast strain expressing a mutant version (UCP⌬9) of the uncoupling protein of brown adipose tissue, when induced with galactose. The expression of this mutant protein produces a complete uncoupling of the mitochondria, thus abolishing the contribution of the mitochondria to the cell fluorescence (18,42). In the first experiment, we grew the cells in 2% glucose or 2% galactose, finding no differences in fluorescence (Table III). In the second experiment, the cells were first grown on lactate (respiratory conditions) and then galactose was added at a concentration (1%) that could be fermented when the mitochondria was not functional. The results showed that the fluorescence of the cells on lactate was reduced to a half upon the addition of galactose (Table III). From these results we concluded that, using 1 nM DiOC 6 (3), the mitochondria did not make any significant contribution to the cell fluorescence in fermenting conditions, and it contributed  50% in respiratory conditions.
The results presented in Fig. 3, A and B, indicated that K ϩ starvation greatly increased the cellular fluorescence, probably reflecting a hyperpolarization of the membrane potential. This hyperpolarization was consistent with the results obtained in N. crassa cells using direct measurements (3), if we assumed that higher fluorescence corresponded to more negative inside membrane potential. To test this directly we measured the cellular DiOC 6 (3) fluorescence in N. crassa, selecting in the flow cytometer the population of germinated conidia which were only slightly larger than S. cerevisiae single cells. Although N. crassa does not ferment, the known contribution of the mitochondria to the fluorescence of S. cerevisiae respirating cells stained with 1 nM DiOC 6 (3) prompted us to perform the test. Giving a 100% value to the cellular DiOC 6 (3), fluorescence of N. crassa normal-K ϩ cells, for which the membrane potential is Ϫ200 mV, the fluorescence of low-K ϩ cells, for which the membrane potential is Ϫ300 mV (3), was 205%. The addition of K ϩ to the low-K ϩ cells of N. crassa, which is known to cause considerable depolarization (3), also produced a remarkable decrease of the cellular DiOC 6 (3) fluorescence, to 141% upon addition of 10 mM K ϩ . All these results supported that the cellular DiOC 6 (3) fluorescence could be used to assess differences in the membrane potential of isogenic strains differing only in genes encoding K ϩ transporters, and that the increase in fluorescence can be taken as hyperpolarization of the plasma membrane.
trk1⌬ Cells Are Highly Hyperpolarized-Flow-cytometric analyses of the cellular DiOC 6 (3) fluorescence in TRK1 TRK2, TRK1 trk2⌬, trk1⌬ TRK2, trk1⌬ trk2⌬, trk1⌬ trk2⌬ HAK1, and trk1⌬ trk2⌬ HKT1 cells, either normal in K ϩ or K ϩ -starved, before and after the addition of KCl or NaCl, revealed that the cellular DiOC 6 (3) fluorescence, and hence the membrane potential, depended on the genotype (Table IV). Comparing first the TRK1 TRK2 and trk1⌬ trk2⌬ strains, it was clear that the latter was hyperpolarized with reference to the former, both in conditions of a normal K ϩ supply and in K ϩ -starved conditions. In K ϩ -starved trk1⌬ trk2⌬ cells, the addition of 10 mM KCl or NaCl produced further hyperpolarization, whereas in TRK1 TRK2 cells NaCl showed no effect and KCl depolarized. Deletion of TRK2 did not produce any significant effect with reference to the wild strain, but TRK1-deleted cells (trk1⌬ TRK2 strain) showed a hyperpolarized state between the TRK1 TRK2 and trk1⌬ trk2⌬ cells. Interestingly, in the trk1⌬ TRK2 strain neither NaCl nor KCl showed any appreciable effect.
The presence of the HAK1 or HKT1 (in the absence of Na ϩ ) genes in the trk1⌬ trk2⌬ strain did not change notably the membrane potential of cells with a normal K ϩ content. In K ϩ -starved cells, the presence of these genes did not change the hyperpolarized state of these cells in basal conditions (absence of K ϩ and Na ϩ ), but changes occurred when these transformants were treated with NaCl and KCl. In HAK1 cells, KCl depolarized and NaCl hyperpolarized, whereas in HKT1 cells both KCl and NaCl depolarized. When the HKT1 transformant was grown in the presence of Na ϩ the cells were strongly depolarized consistent with the high tolerance of this transformant to hygromycin B (YPD contains both K ϩ and Na ϩ ).
Considering that Trk1p and Hak1p are very active K ϩ transporters, that Hkt1p is a K ϩ -Na ϩ transporter, which may also transport only Na ϩ in some conditions (34), and that Trk2p has negligible activity as a K ϩ transporter in the strains used, the most important conclusions that can be drawn from the results summarized in Table IV are the following: (i) both Trk1p and Trk2p are involved in the control of the membrane potential, even when neither K ϩ nor Na ϩ are present in the external medium; (ii) the heterologous transporters did not suppress the high membrane potential of trk1⌬ trk2⌬ cells in the absence of K ϩ and Na ϩ ; (iii) electrophoretic K ϩ and Na ϩ uptake can explain the depolarizing effects shown by KCl and NaCl in those cells expressing an active K ϩ or Na ϩ transporter; (iv) in the absence of Trk1p and Trk2p, KCl and NaCl showed hyperpolarizing effects, especially in K ϩ -starved cells.
Chloride Is Not Involved in the Hyperpolarizing Effects of NaCl and KCl-One intriguing result among those summarized in Table IV is the hyperpolarizing effect of KCl and NaCl in the trk1⌬ trk2⌬ strain. This effect could result from the high diffusion potential created by the increase in the external concentration of chloride if a chloride channel exists in the plasma membrane. To address this possibility we repeated the experiments summarized in Table IV substituting Na ϩ -MES and K ϩ -MES for NaCl and KCl, respectively. The change of the anion did not change the results, thus indicating that Na ϩ and K ϩ , and not chloride, were involved in the hyperpolarizing effect.
Trk1p and Trk2p Are Involved in the Response of the Membrane Potential to Changes in the External pH-Two reasons could explain why trk1⌬ trk2⌬ cells were hyperpolarized with reference to TRK1 TRK2 cells, that the H ϩ -pump ATPase was more active in the mutant than in the wild strain, because Trk1p and Trk2p exercise some degree of control on the pump, or that Trk1p and Trk2p depolarized the membrane by mediating a flux of ions at high membrane potentials. Because the difference between the membrane potentials of these strains persisted in a buffer with only MES, Ca 2ϩ , and H ϩ , the most likely ion fluxes were H ϩ inward or anions outward. To test if H ϩ influx was involved, we studied the response of the cellular DiOC 6 (3) fluorescence of the different genotypes to changes in the external pH and to the addition of uncouplers. These tests were performed with K ϩ -starved cells in the complete absence of K ϩ , thus eliminating the differences in depolarizing currents that this cation may introduce in genotypes with different K ϩ uptake capacities. The data summarized in Table V (data at pH 6.0 correspond to basal conditions, K ϩ -starved cells, in Table  IV) show clearly that the membrane potential decreased with the decrease of the external pH only when Trk1p or Trk2p are present. In contrast, the response to the addition of CCCP, which should increase unspecific H ϩ conductance, was not appreciably different in the different genotypes, producing in all cases a progressive decrease in the cellular DiOC 6 (3) fluorescence with the increase in the CCCP concentration. Taken together, these results suggest that Trk1p and Trk2p somehow increase the permeability to H ϩ .
Nitrogen Starvation Also Produces Hyperpolarization-Before addressing the question of whether Trk1p and Trk2p are H ϩ transporters or only regulate the H ϩ permeability of another membrane protein, we addressed the more general question of whether hyperpolarization of the plasma membrane was a specific response to K ϩ starvation or a more general response to any kind of starvation. With this purpose we studied the cellular DiOC 6 (3) fluorescence of nitrogen and phosphatestarved cells, finding that fluorescence increased as a consequence of nitrogen starvation but not after phosphate starvation. We have described that trk1⌬ trk2⌬ cells hyperpolarized in response to K ϩ starvation, increasing their cellular DiOC 6 (3) fluorescence over the already high normal value. Unlike this response, nitrogen starvation did not show any effect on the DiOC 6 (3) fluorescence of trk1⌬ trk2⌬ cells. DISCUSSION The results presented in this report indicate that the growth of trk1⌬ trk2⌬ mutants of S. cerevisiae is supported by an ectopic K ϩ uptake mediated by several transporters, which, as a whole, resembles a low-affinity single transporter for K ϩ , Rb ϩ , Na ϩ , and Li ϩ . The view is supported by the manner in which this low-affinity uptake is inhibited by ammonium and by divalent cations (Fig. 1), and by the number of different and unrelated transporters (amino acids, sugar, inositol, and choline transporters) (this report and Refs. 14 -16) that by overexpression partially suppress the defective growth of trk1⌬ trk2⌬ strains at low K ϩ . Our results also indicate that in the absence of Trk1p, or Trk1p and Trk2p, the membrane potential is exceptionally high, negative inside. Taken together, these results pose two questions, whether there is any causal relationship between the ectopic low-affinity K ϩ and Rb ϩ uptake and the high membrane potential, and what the cause of this high membrane potential is? Regarding the former question, it is clear that both the low-affinity Rb ϩ uptake and the high membrane potential are the consequences of the deletion of the TRK1 gene. Furthermore, the ectopic K ϩ uptake must result from a change in a general property of the membrane, because it is unlikely that the deletion of the TRK1 gene can modify the specific properties of many different transporters, making all of them permeable to alkali cations. Considering these observations and the high sensitivity of the low-affinity Rb ϩ uptake to CCCP, the most likely cause for the ectopic uptake of K ϩ is the unusually high membrane potential exhibited by trk1⌬ strains.
Regarding the high membrane potential of trk1⌬ and trk1⌬ trk2⌬ cells, our results reveal that Trk1p and Trk2p are not only K ϩ transporters but also essential regulators of the membrane potential. To fulfill this function they must either counter the activity of the pump or control the conductance of the plasma membrane. Before going further in the discussion of the function of Trk1p and Trk2p, it is worth mentioning that a pitfall in the use of the cellular fluorescence of the DiOC 6 (3) probe to assess differences in membrane potentials is extremely unlikely, considering the conditions for our assessments: (i) we used the cellular DiOC 6 (3) fluorescence only for comparative purposes in entirely isogenic cells, except for the genes considered in each case; (ii) the contribution of the mitochondria to these changes in fluorescence has been ruled out; (iii) we did a control using N. crassa cells, for which the membrane potential has been measured using intracellular electrodes (3). Furthermore, the hyperpolarized state of the trk1⌬ and trk1⌬ trk2⌬ cells can be also deduced by contrasting the phenotype of these mutants with the phenotype of pma1 mutants. The latter are more resistant to hygromycin B than the wild type, whereas trk1⌬ and trk1⌬ trk2⌬ mutants are strikingly more sensitive (Table II). Assuming that the phenotype of pma1 mutants is the result of a lower membrane potential (32), it can be concluded that the phenotype of trk1⌬ and trk1⌬ trk2⌬ mutants is the result of a higher membrane potential.
Nitrogen starvation hyperpolarized TRK1 TRK2 cells but not trk1⌬ trk2⌬ cells, whereas K ϩ starvation hyperpolarized both types of cells. This suggests that at least two parallel and additive routes control the membrane potential, one dependent on Trk1p and Trk2p, and the other independent of these proteins. Apparently, K ϩ starvation activates both routes, whereas nitrogen starvation activates only that dependent on Trk1p and Trk2p. The mechanisms involved in the control of the membrane potential cannot be established at this moment. However, there are only two ways to achieve this control, either modifying the activity of the pump or triggering a "safety a K ϩ contents in the growing media were: 0.5 mM for TRK1 TRK2, TRK1 trk2, trk1 trk2 HAK1, and trk1 trk2 HKT1, 2.5 mM for trk1 TRK2, and 10 mM for trk1 trk2 strains. b trk1 trk2 HKT1 /Na ϩ cells were grown in the arginine medium supplemented with 0.5 mM NaCl and 0.5 mM KCl and suspended in testing buffer containing 0.5 mM Na ϩ and 0.5 mM K ϩ . K ϩ -starved cells were prepared following the standard method, and then suspended in testing buffer supplemented with 0.5 mM NaCl. a Cells were grown as stated in Table IV and charged with DiOC 6 (3) in the testing buffer (pH 6.0), following the standard method; then, tartaric acid, TAPS, or CCCP were added to reach the selected pH or the reported concentration of CCCP. Measurements were made 60 to 90 s after the addition.
depolarizing current" when the membrane potential reaches a certain value. In the latter case, because the function can be performed in the absence of K ϩ , or any other alkali cation (Table IV), it is likely that the ions moving are either H ϩ inward or anions outward. The Trk1p-Trk2p dependence of low-pH depolarization (Table V) indicates that an inward movement of H ϩ may be involved in the route of control depending on Trk1p and Trk2p.
The capacity of Trk1p and Trk2p to control the membrane potential is not a general property of K ϩ transporters, because Hak1p cannot perform these functions (Tables III and IV). Although the results supporting this conclusion have been obtained using a heterologous expression, the high level of conservation in proteins and functions among fungi, and even among fungi and higher plants, suggests that it is correct. It is very interesting that in S. occidentalis, 2 in N. crassa, 3

and in
Debaryomyces hansenii 4 HAK1 type K ϩ transporters coexist with TRK2 K ϩ transporters. Whether the function of these TRK2 transporters in these species is more related with the control of the membrane potential than to K ϩ uptake is now under study. Interestingly, in barley plants K ϩ uptake is mediated by HAK1 transporters (43)(44)(45)(46), and wheat plants have a HKT1 transporter whose function is not clear (47). Hkt1p shows homology with Trk1p and Trk2p (36) and, when expressed in S. cerevisiae, it produced strong depolarization in the presence of Na ϩ (Table IV). Therefore, the involvement of Hkt1p in the control of the membrane potential in higher plants is an attractive possibility.
The conclusion of this and previous reports (14 -16) identifying independent mechanisms for Rb ϩ influx in TRK1 TRK2 and trk1⌬ trk2⌬ cells indicates that the mechanisms involved in the activation of the V max of Rb ϩ influx by glucose in both types of cells (17) may be also different. Interestingly, we found that the addition of glucose hyperpolarized both wild type and trk mutant cells (not shown), which is consistent with the known activating effect of glucose on the H ϩ -pump ATPase (48). The possibility that this hyperpolarization brings about the increase of the V max of Rb ϩ influx in all cases is an attractive idea. Unfortunately, this cannot be tested with the techniques used in this report.
Finally, trk1⌬ trk2⌬ mutants have been extensively used for cloning heterologous K ϩ -transport genes. Our results indicate that genes expressing low-rate, low-affinity K ϩ uptake in these mutants, may encode non-K ϩ transporters that support ectopic K ϩ uptake.


extfieldsofstudy: Biology Medicine

text:
Functional Characterization and Localization of Protein Phosphatase Type 2C from Paramecium *

We cloned a protein phosphatase 2C gene fromParamecium (PtPP2C), which codes for one of the smallest PP2C isoforms (Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L. A., and Schultz, J. E. (1994)J. Biol. Chem. 269, 32774–32780). After mutation of 9 ciliate Q codons (TAA) to CAA PtPP2C was expressed as an active protein in Escherichia coli. The catalytic core region contains 284 amino acids as defined by C- and N-terminal deletions. The C terminus from amino acid 200–300 of PP2C isoforms has only about 20% similarity. To demonstrate that the carboxy end is in fact needed for activity, we generated an enzymatically active PtPP2C containing a C-terminally located tobacco etch virus-protease site. Upon proteolytic truncation enzyme activity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously identified to be essential for PP2C catalysis. Mutation of the hydrophobic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile214 is in a loop region without defined secondary structure, our data clearly go beyond the x-ray structure. The functional equivalence of the 180 amino acid long C terminus from the bovine PP2C with the 100 amino acid long carboxy end of the PtPP2C was demonstrated by producing an active chimera, i.e. the PP2C from Paramecium has no obvious regions which may be specifically involved in subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold labeling in the cytosol and nucleus and very distinctly on the ciliary microtubule/dynein complex. The data suggest a role for PtPP2C in the regulation of dyneins, i.e. in cellular cargo transport and ciliary motility.

Recently, substantial progress has been made in the area of serine/threonine protein phosphatases. Recombinant type 1 protein phosphatase (PP1) 1 ␣ and ␤ isoforms and PP2B, members of the same, highly conserved PPP protein phosphatase family, have been crystallized (1)(2)(3)(4). In addition, several physiological functions of PP1, 2A, and 2B have been tentatively identified using specific inhibitors (for a review, see Cohen (5)). No specific inhibitors are available for protein phosphatase 2C isozymes (PP2C) which belong to a separate family of Mg 2ϩ -or Mn 2ϩ -dependent phosphatases (PPM-family) with a low degree of conservation in their primary structure (6,7). The molecular weights range from 48 kDa (from Arabidopsis thaliana; 433 amino acids) to the smallest versions of 33 kDa from Paramecium tetraurelia (300 amino acids) and 31 kDa from Saccharomyces cerevisiae (TPD1, 282 amino acids) (8). This large variability was suggested to indicate a functional diversity of PP2C enzymes although presently our knowledge about PP2C functions is rather limited (9). Lately, a deletion analysis with a human liver PP2C␣ isoform (382 amino acids) was reported (10). Two C-terminal truncations of 75 and 179 and one Nterminal deletion of 51 amino acids were constructed. Only the expression product of PP2C␣1-307 was active. This is in agreement with the recently published x-ray structure of a human PP2C␣, which shows that the C terminus starting around amino acid position 300 contains three ␣ helices forming an appendix to the catalytic core (9). Consequently, the C-terminal domain was speculated to affect substrate specificity. The 300amino acid long protozoan PP2C completely lacks this C-terminal domain. How are the cellular localization and substrate specificity of PP2C defined in the ciliate? First, we determined the minimal core structure of the PtPP2C, which is essential for catalytic activity, i.e. we constructed N-and C-terminal deletion mutants at the DNA level and, additionally, C-terminal truncations via targeted proteolysis. Fortuitously, we identified an invariant isoleucine at position 214 in an unordered loop region as cardinal to enzyme activity in PP2C enzymes. Second, we assessed the functional equivalence of protozoan PP2C regions with their mammalian counterparts by generating an active bovine/ciliate chimera. Finally we localized PP2C in Paramecium by immunochemical and -cytochemical methods in the cytoplasm, nucleus and the cilia. Because of the targeting of PtPP2C to motor components in the cilia we propose that PtPP2C participates in the regulation of cytoplasmic, nuclear and ciliary dynein regulation. site-directed mutagenesis (11). NdeI and XhoI restriction sites were added by PCR to the 5Ј and 3Ј ends, respectively, to clone the mutated PtPP2C cDNA into the pET16b expression vector.
Construction of a Ciliate/Bovine PP2C Chimera-The cDNA for the bovine PP2C␣ isoform with 5Ј-XhoI and 3Ј-BamHI restriction sites was from Dr. D. Selke, University of Marburg. Using the PtPP2C as the N-terminal region a PCR fragment was generated which had an MaeI 3Ј-restriction site at base pairs 578, i.e. at G193 within the third conserved region (12) and an NdeI cutting site for ligation into pET16b at the 5Ј-end. The complementary C-terminal fragment of the bovine PP2C␣ had an MaeI site 5Ј and an XhoI site 3Ј. The appropriately digested and gel-purified PCR products and NdeI/XhoI cut pET16b were used in a triple ligation to obtain the full-length chimeric PP2C DNA construct in the expression vector.
Deletions-At the N terminus all deletions retained the methionine at position 1 as part of the NdeI restriction site. The N-terminal truncations were PtPP2C⌬2-3, PtPP2C⌬2-11, PtPP2C⌬2-13, PtPP2C⌬2-15, and PtP2C⌬2-19. Truncations at the C terminus were made by cutting off amino acids one by one up to a total of 10. All constructs were generated by PCR using respective oligonucleotide primers and plasmid pET16b/PtPP2C as a template. The PCR products were cloned into the NdeI/XhoI sites of the pET16b expression vector.
Insertion of Protease Digestion Sites-A TEV protease digestion site (ENLYFQG) was introduced into PtPP2C at positions 219 and 254. Because the DNA sequence encoding the TEV cleavage site can be made to contain an EcoRII restriction site (GAGAATCTTTATTTCCAGGGC) which is absent in the PtPP2C cDNA, placement of the TEV sites could be freely selected. We replaced amino acids 219 -226, PtPP2C 219TEV , and 254 -261, PtPP2C 254TEV HisN. To switch the His tag in PtPP2C 254TEV HisN to the C terminus creating PtPP2C 254TEV HisC, we recloned PtPP2C 254TEV into the NcoI site of pET16b in front of the His tag. A Factor Xa cleavage site (IEGRA), whose encoding cDNA contained an AscI restriction site (ATCGAAGGGCGCGCC), was introduced into the PtPP2C by replacement of the amino acids 212-216, PtPP2C 212FXa . Single amino acid exchanges at positions 212 to 215 are listed in Table I. The fidelity of all constructs was verified by DNA sequencing.
DNA Primers-A list of the primers used in this study is available from J.E.S.
Expression and Purification-Usually, single transformed colonies of E. coli BL21DE3plysS were inoculated into 5 ml of LB medium containing 50 g/ml ampicillin and 34 g/ml chloramphenicol and grown over night at 37°C and 260 rpm. 400 l were used to inoculate 20 ml of LB medium with antibiotics, grown to an A 600 of 0.4 -0.6 and induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside. After 3 h, cells were harvested (3,200 ϫ g, 4°C, 10 min) and resuspended in 800 l of binding buffer (20 mM Tris/HCl, 5 mM imidazole, 250 mM NaCl, pH 7.9). Bacteria were broken by freeze-thaw and sonication and centrifuged (10,000 ϫ g, 4°C, 20 min). 100 -300 l of the supernatant were applied to a 25-l NiNTA column equilibrated with binding buffer. The column was washed with 250 l of binding buffer and 150 l of wash buffer (20 mM Tris/HCl, 60 mM imidazole, 500 mM NaCl, pH 7.9). Bound protein was eluted with 150 l of elution buffer (20 mM Tris/HCl, 1 M imidazole, 500 mM NaCl, pH 7.9). Protein purity was monitored by SDS-polyacrylamide gel electrophoresis stained with Coomassie Brilliant Blue. Protein concentration was determined by the Bio-Rad method. The conditions for TEV digestion were as suggested by Life Technologies, Inc.
PP2C Assay-Bovine casein (Sigma C-4765) was phosphorylated with [␥-32 P]ATP by cAMP-dependent protein kinase and purified according to McGowan and Cohen (13). PP2C assays (30 l) contained 20 mM Mg 2ϩ acetate, 50 mM Tris/HCl, pH 7.0, 0.1% 2-mercaptoethanol, 0.6 mg/ml bovine serum albumin, and 1 M [ 32 P]casein (about 30,000 cpm). After 10 min at 30°C dephosphorylation was stopped by addition of 200 l of 20% trichloroacetic acid (w/v). After centrifugation radioactivity was determined in the supernatant. Dephosphorylation was restricted to 30% of the total 32 P activity to guarantee linearity. One unit of activity is the amount of enzyme which catalyzes the dephosphorylation of 1 mol of [ 32 P]casein/min (13).
Cell Fractionation for Immunohistochemical Analysis-Paramecium 7S wild-type cells, grown to early stationary phase in sterile medium (16), were harvested and deciliated by a CaCl 2 shock (10 min on ice in 5 mM Pipes/HCl, pH 7.0, 3 mM KCl, and 40 mM CaCl 2 ). Cell fractionation, protein determination, and enzyme-linked immunosorbent assays were carried out as described earlier (17).
For post-embedding labeling cells were fixed for 10 min at 22°C in 4% formaldehyde ϩ 0.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1. After ethanol dehydration, cells were impregnated with Unicryl (British BioCell Int., London) according to the manufacturer's instructions. Ultrathin sections were prepared, labeled with pA-Au 6 , stained, and quantitatively evaluated as described elsewhere (18).

RESULTS
Expression and Characterization-We cloned a PP2C gene from a Paramecium cDNA library (12). Its heterologous expression was impossible because in this protozoan the universal stops TAA and TAG code for Q. We now mutated all 9 TAA/Q triplets of the PP2C cDNA to universal CAA/Q triplets and inserted the gene into the expression vector pET16b, thereby attaching a deca His tag and a factor Xa protease site to the N terminus. PtPP2C was expressed in E. coli BL21(DE3)plysS. Approximately 8 mg of enzymatically active PtPP2C (about 30 milliunits/mg) were purified from a 1-liter culture, i.e. the high A/T content of the Paramecium gene (62%) and the resulting skewed codon usage did not affect the expression in E. coli which has an A/T content of only 50%. A comparison of the enzymatic properties of the native, ciliary and the recombinant PtPP2C showed identical temperature optima (46°C), yet, the activation energies derived from linear Arrhenius plots were 69 and 30 kJ/mol for the ciliary and the recombinant enzyme, respectively. The most prominent difference was the effect of divalent cations. Substituting 20 mM Mg 2ϩ with 20 mM Mn 2ϩ decreased the activity of the native enzyme by 36%, whereas the activity of the recombinant enzyme was almost doubled. Posttranslational modifications of the ciliary enzyme may account for these differences.
Deletion Analysis-With only 300 amino acids, the PtPP2C is one of the shortest enzymes of the PPM family. To determine the amino acids essential for catalytic activity we constructed and expressed enzyme proteins which were truncated either at the 5Ј-or the 3Ј-end. At the N terminus the following amino acid deletion mutants were generated: PtPP2C⌬2,3, PtPP2C⌬2-11, PtPP2C⌬2-13, PtPP2C⌬2-15, and PtPP2C⌬2-19. All constructs were expressed as soluble proteins with approximately equal efficiency (Fig. 1A). Removal of up to 10 N-terminal amino acids affected enzymatic activity only slightly. However, deletion of 12 N-terminal amino acids (PtPP2C⌬2-13) diminished activity by 80%. Larger N-terminal truncations yielded soluble expression products, albeit without activity (Fig. 1A).
At the C terminus 4 amino acids (YLRR) were sequentially removed one by one without loss of activity, e.g. the specific activity of PtPP2C⌬297-300 was comparable to the full-length enzyme (Fig. 1B). The expression of these constructs was about as good as the untruncated PtPP2C. Removal of 5 (VYLRR) or more amino acids (up to 10) from the carboxy end led to drastic changes. First, enzyme activity was lost; second, the yield of purifiable material was diminished, i.e. either the expression rates or the stability of the expressed proteins was reduced.
Notably, all C-terminally shortened expression products displayed an anomalous electrophoretic mobility upon SDS-polyacrylamide gel electrophoresis, running at an apparent larger molecular mass than the untruncated version ( Fig. 1B and data not shown). Therefore, in addition to the routine verification of all constructs by DNA sequencing we used amino acid analysis and ion-spray mass spectrometry to ascertain the identity of PtPP2C⌬299 -300. Data from both methods unequivocally established the correct identity (data not shown). The anomalous electrophoretic mobility of the shortened expression products may be related to the removal of the two terminal basic arginine residues.
C-terminal Truncation by Proteolysis-PP2C isoforms from various phyla share three reasonably long conserved regions within the 200 N-terminal amino acids and only very short stretches of conservation from 200 to 300 (6,12). Therefore, we reasoned that the highly diverged C termini may partly be dispensable for catalysis but serve other roles, e.g. in protein folding, targeting to specific cellular locations, or substrate binding. To test this hypothesis we inserted at two different positions a TEV-protease recognition site, which should permit a C-terminal truncation by proteolysis after expression, folding, and purification. Amino acids 219 -226 (PtPP2C 219TEV , replacement of the weakly conserved sequence DVKKTEL in the ␤9 strand) and 254 -261 (PtPP2C 254TEV HisN, replacement of the nonconserved TIGQAQV in the ␣5 helix) were changed to the TEV cutting sequence ENLYFQG. The constructs were expressed as soluble proteins, purified by NiNTA chromatography, and were recognized by a PtPP2C-specific antibody in Western blots at their calculated molecular masses (data not shown).
PtPP2C 219TEV was inactive. To reduce the number of ex-changed amino acids as well as to replace less conserved amino acids we created PtPP2C 212FXa in which the sequence QLII (212-215) was changed to IEGR, thus generating, in conjunction with Ala 216 , a factor Xa cleavage site (IEGRA). Somewhat surprisingly, PtPP2C 212FXa was also inactive (Table I). Individual amino acid exchanges at each of the changed positions showed that exclusively the I214G conversion was responsible for the loss of function (Table I). In an I214A PtPP2C expression product 62% of wild-type activity was retained, and an I214L PtPP2C was at least as active as the wild-type enzyme (Table I). Strikingly, the adjacent isoleucine at position 215 was not crucial for enzyme activity. Neither an I215R nor an I215G conversion abolished PtPP2C activity (Table I).
The highly specific knock-out of the enzymatic activity by the replacement of Ile 214 with G was unexpected because in the x-ray structure of the human PP2C␣ isoform the corresponding region is without a defined secondary structure (9). Therefore, we wanted to know whether the result with the PtPP2C was due to a structural peculiarity of the protozoan enzyme or was representative of an important, as yet unrecognized structural feature common to all members of this class of phosphatases. Alignment shows that in the bovine PP2C␣ isoform a valine at position 215 corresponds to Ile 214 of the PtPP2C (see Fig. 3 for an extended alignment of PP2C sequences). A bovine PP2C␣ construct in pPET16b, which contained a V215G conversion, was expressed in E. coli, yet it was enzymatically inactive, whereas the expressed Val 215 wild-type enzyme was active (Table I).
PtPP2C 254TEV HisN carries the TEV site in a linker region connecting the ␣5 and ␣6 helices. The expressed protein was active, i.e. the nonconservative exchange of 6 amino acids in   this region did not impair protein folding or phosphatase activity (Fig. 2). Upon TEV digestion enzymatic activity was strongly reduced. Yet, due to the incomplete proteolysis and an apparent association of the digested C-terminal peptide with the remaining protein we were unable to fully separate PtPP2C⌬254 -300 from the 40-amino acid proteolytic fragment as apparent in Western blots (data not shown). Therefore, we switched the His tag to the C terminus (PtPP2C254 TEV HisC). This did not affect phosphatase activity. Now, after TEV digestion we obtained pure PtPP2C⌬254 -300 in the flow-through of a NiNTA column which retained all undigested protein and the C-terminal peptide (Fig. 2). PtPP2C⌬254 -300 was devoid of phosphatase activity. We conclude that the complete C terminus of the PtPP2C with the exemption of the last 4 amino acids is required for catalytic activity. Taken together the truncation experiments define a PtPP2C of 284 (13-296) as the catalytic core.
PP2C Chimera-To prove the functional interchangeability of the C termini we constructed a hybrid PP2C enzyme in which the conservative N terminus (amino acids 1-193) from Paramecium was linked within the conserved region III ( 193 SRALGD 198 ) to the C-terminal end from the bovine PP2C␣, i.e. 188 amino acids (195-382) of the mammalian enzyme replaced 105 amino acids (194 -300) from the PtPP2C. This chimera was expressed as a soluble protein and had a specific activity of 14 milliunits/mg, roughly as the parent PP2C isoforms (35 and 8 milliunits/mg for the Paramecium and bovine PP2C, respectively). This demonstrated that the N and C termini of the bovine and protozoan PP2C are functionally interchangeable despite the low degree of sequence conservation at the amino acid level (22%). Furthermore, it showed that the 80-amino acid extension of the bovine PP2C is irrelevant as far as enzyme activity is considered.
Cellular Localization of PP2C in Paramecium-All PP2C proteins lack pre-or prosequences and nothing is known about secondary modifications which may serve to target the protein to a particular cytoplasmic compartment. Based on the above data, we must assume that in the PtPP2C only 12 N-terminal and possibly four C-terminal amino acids may be used for subcellular targeting or substrate recognition. It is the more important to determine whether the PtPP2C shows a distinct subcellular localization in the ciliate, which might lead to clues concerning physiological functions of this enzyme. PtPP2Cspecific enzyme-linked immunosorbent assays of different cellular fractions showed that 98.6% of the immunoreactivity reside in the deciliated cell bodies, 0.8% in the isolated cilia and 0.6% in the supernatant of the deciliation procedure. Considering that the volume of the cell body is about 98% and of the cilia 2%, one can assume that the PP2C concentration in these compartments is similar. As seen in immunofluorescence analysis of permeabilized cells (Fig. 4A), cilia contained substantial amounts of PP2C.
A more detailed immunocytochemical analysis was carried out by post-embedding pA-Au 6 labeling which avoids loss of soluble proteins (Fig. 4 and Table II). Labeling density in the cytoplasm, including the endoplasmic reticulum, exceeded that in any other organelles. Only the nuclei and the cilia were also labeled, whereas other identifiable structures were not significantly labeled (Fig. 4B). Within the cytoplasm a differentiation between cytosol and endoplasmic reticulum was not possible. Within the cilia, a more detailed structural analysis was made using pre-embedding labeling with pA-Au 6 . In this method access of the antibody is facilitated by the presence of 0.05% Triton X-100 as a detergent during fixation. This method could be used because biochemical data have shown that in the cilia the PtPP2C is in the particulate fraction and requires 1% Brij 35 as a detergent for solubilization (12). We observed that 83% of the gold label was associated with axonemal structures, i.e. with the microtubules (42%) and dynein arms (26%) and only Identical amino acids are in boldface type. The human PP2C␥ has a long insert of unknown function at the N terminus, which is responsible for the skewed numbering compared with the other isoforms.
3% with the ciliary membrane (Table III). In agreement with this it was found that horseradish peroxidase labeling was restricted to the axonemes (Fig. 4C).

DISCUSSION
With a length of 300 amino acids the PtPP2C is short compared with most mammalian isoforms, which are between 380 and 410 amino acids long (6). The PtPP2C sequence mainly comprises those regions that form the catalytic core. In particular, it lacks the mammalian C-terminal domain of 80 -100 amino acids, which is placed as an appendix apart from the catalytic site. This C-terminal extension is hypothesized to be involved in substrate recognition and specificity (9). Other PP2C catalytic domains occur in a variety of different structural contexts, which probably mediate interactions with particular substrates (6). In the short PtPP2C, substrate recognition and thus functional unambiguity must either reside within its 300 amino acids or be determined by a specific subcellular localization. We approached this question in three ways, (i) definition of regions within the PtPP2C not essential for enzyme activity, (ii) demonstration of the interchangeability of the highly diverged C-terminal domains, and (iii) immunocytochemistry.
At the N terminus the first inactive deletion mutant was PtPP2C⌬2-15. This truncated enzyme lacks 6 (RDKTTT) of the 11 amino acids which assemble into the ␤1 domain. Although the ␤1 region does not itself participate in catalysis, the loss of it probably destabilizes the central ␤ sandwich which serves as a scaffold for the active site (9). At the C terminus, removal of only five amino acids resulted in an inactive protein. This is most plausibly explained by the fact that the ␤11 strand is affected, which connects into the active site and stabilizes the conserved Asp 289 , one of the amino acids of the metal-binding site. Thus, the minimal sequence required for formation of a catalytically active entity seems to be a 284-amino acid long protein, PtPP2C  . The truncation data support the notion that the three-dimensional structure of the PtPP2C probably is more or less identical to that determined with the recombinant human PP2C␣ despite the overall poor amino acid conservation (31%).
To unequivocally demonstrate that the C-terminal end of PtPP2C is indispensable for activity, we created mutants that could be proteolytically truncated after expression and folding. A nonconservative 7-amino acid replacement at position 254 with a TEV-protease site resulted in formation of an active protein. The truncation by TEV showed that the ␣5 helix on the surface of the enzyme is accessible to proteolysis. The digested protein was inactive. This means that the ␣5, ␣6-helices and the ␤11 strand are cardinal structural elements for enzyme activity. In PtPP2C 219TEV 7 amino acids in the ␤9 strand were exchanged. Obviously, the ␤9 strand was affected so profoundly that an inactive protein resulted. Therefore, a factor Xa cutting site was introduced into an unordered loop region in front of the predicted ␤9 strand. Surprisingly, PtPP2C 212FXa was also inactive. This observation compellingly argued that the unordered loop region plays a completely unsuspected role in catalysis. Individual permutations at all positions involved identified Ile 214 to be most important for activity (see Table I). Indeed, a sequence comparison of eukaryotic PP2C isoforms showed that in the equivalent position there is always an I or V at an invariant distance of 15 amino acids from a conserved RXXGD sequence and four amino acids in front of the ␤1 region (Fig. 3). The latter invariant amino acids are still in the loop region which has no secondary structure (9). Because the I214A mutant had 60% of the wild-type activity and an I214L mutant was fully active, we conclude that the steric bulk of Ile 214 (PtPP2C numbering) is needed to sustain a catalytically competent conformation when phosphocasein is used as a substrate. This point was further strengthened by the lack of activity of a corresponding mutation in the bovine PP2C␣. In this respect, our data clearly go beyond the picture which has emerged from the x-ray structure of PP2C (9). It does not show why a mutation that replaces Ile 214 in PP2C results in inactivation. The loss of a hydrophobic side chain must alter the stability or a directed movability of this part of the protein such that the active site centers cannot stabilize an enzyme/substrate configuration involved in a stabile transition state, i.e. Ile 214 may be involved in substrate recognition and specification. The sequence comparison (Fig. 3) identified position 215 (PtPP2C numbering) as variant (Fig. 3) thus explaining why amino acid replacements of Ile 215 were immaterial. On the other hand it is notable that the invariant Gln 212 could be replaced by I without affecting enzyme activity.
The C-terminal domains of PP2C, which start around position 200, are characterized by a particularly high extent of divergence (up to 80%). This region might, therefore, participate in the definition of substrate specificity as a secondary function. We tested the functional equivalence of the C termini by generating a PP2C chimera which consisted of PtPP2C1-193/bovine PP2C194 -382. The full functional replacement of the carboxyl-terminal 100 amino acids from the PtPP2C by the very poorly conserved 184 amino acids from of the bovine PP2C␣ demonstrated that quite extensive alterations in the primary amino acid sequence at the carboxy end are tolerated without dramatically affecting enzyme activity. This finding strengthens the assumption that the C-terminal end of mammalian PP2Cs is not even peripherally involved in catalysis and has other functions like subcellular targeting or substrate recognition.
In an attempt to reveal clues to a physiological function we established the subcellular localization of PtPP2C in the ciliate. Biochemically, the major fraction of the PtPP22C is cytosolic, a minor part is structurally associated with the cilia (12). Now we combined partial cell-fractionation and enzyme-linked immunosorbent assay with (ultra-)structural immunolabeling techniques. Considering the soluble cytosolic PtPP2C we used postembedding immunogold electron microscopy techniques and with the cilia additionally preembedding labeling.
The most distinct subcellular localization of PtPP2C was found in the cilia, where it was associated with microtubules and dynein, i.e. it was clearly targeted to the ciliary motor (Table III), implicating a motor component as a PtPP2C substrate. This peculiar ciliary localization may be helpful when discussing the cytoplasmic and nuclear localization of the PP2C reported here. A family of cytoplasmic dyneins exists, which in association with dynactin and microtubules are major players in the transport of cellular cargo such as membraneous organelles, possibly large protein complexes and mRNA. This dynein-motor complex can be regulated by phosphorylation and dephosphorylation (for a recent review, see Hirokawa (19)). Indeed, Schroeder et al. (20) have demonstrated the presence of a microtubule-based motor in the cell body of Paramecium, which in association with a cytoplasmic dynein is involved in vesicle transport. It seems reasonable to consider that the cytoplasmic and nuclear localization of PtPP2C may actually correspond to cellular transport complexes in the ciliate. In agreement with this view is the recent identification of the gene for a PP2C catalytic domain in the vicinity of a cytoplasmic dynein light chain in Drosophila (21). This possibly points to a functional coupling of the gene products. Also recently, PP2C was identified immunocytochemially in the bovine rod outer segment, which is separated from the inner segment by the connecting cilium (22). All metabolic traffic between both segments must run through this structural bottleneck. The PP2C identified in the outer segments may in association with microtubular structures and further motor constituents have a function in this process. The co-localization of dynein and PP2C in the cilia of Paramecium not only would support such a notion, but also identifies this interaction as a meaningful future experimental target. Finally, the structural features encoded in the small PP2C from Paramecium are sufficient to sustain activity and to specifically interact with motor components. The large C-terminal, which is present in so many mammalian isoforms and has no known function, is not required in the ciliate.


s2fieldsofstudy: Biology Medicine

text:
Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production.

Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor kappaB (NF-kappaB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor-associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1-mediated activation of JNK, p38, and NF-kappaB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.

IRAK and IRAK-2 are homologous to Pelle, a Drosophila protein kinase identified genetically to be important in dorsal-ventral pattern formation and in pathogen resistance (22,23). Pelle is essential for the activation of Dorsal, an NF-B-like protein which is mediated by Toll, an IL-1 2074 IRAK Requirement for Optimal Interleukin 1 Signaling receptor homologue in Drosophila (24). The rapid IL-1-dependent association of IRAK and IRAK-2 with the IL-1 receptor complex and their homology to Pelle suggest that IRAK and/or IRAK-2 may serve important functions in initiating IL-1 signaling. However, the roles of IRAK and IRAK-2 in activation of the multiple downstream IL-1 signaling pathways have not previously been determined.
To dissect the role of IRAK in IL-1 signaling pathways, we disrupted the IRAK gene by homologous recombination and prepared IRAK-deficient fibroblasts. IL-1-induced activation of JNK, p38, and transcription factor NF-B, and subsequent induction of IL-6 was analyzed in IRAK-deficient cells.

Materials and Methods
Preparation of IRAK Antibody. Polyclonal rabbit antiserum to IRAK was raised against a peptide (Bio-Synthesis, Inc., Lewisville, TX) corresponding to the COOH-terminal amino acids (657-677) of mouse IRAK protein (25).
Disruption of the IRAK Gene and Generation of IRAK-deficient Mice. Mouse IRAK clones were isolated from a 129/Ola mouse genomic library. A 7-kb BamHI-EcoRI DNA fragment covering the first 9 exons of the mouse IRAK gene was used to prepare the knockout construct. A cassette containing a neomycin resistance gene was used to replace a 940-bp region covering exon 5 to exon 7 of the gene. A herpes simplex-thymidine kinase cassette was placed at the 3 Ј end of the construct. The DNA construct was introduced into E14 embryonic stem cells by electroporation. Cells were cultured in the presence of 400 g/ml G418 and 0.2 M gancyclovir. Embryonic stem cells with the disrupted gene were detected by PCR and then confirmed by Southern hybridization using a DNA probe flanking the 3 Ј end of the construct. Chimeric mice were generated from embryos injected with embryonic stem cells. Germline mice were obtained from breeding of chimeric male mice with C57BL/6J females. Germline female mice heterozygous for the disrupted IRAK gene were identified by PCR. IRAK-deficient male mice carrying only the disrupted IRAK gene were obtained from cross-breeding of heterozygous female mice with wild-type littermates.
Preparation of Embryonic Fibroblasts and Skin Fibroblasts. To prepare embryonic fibroblasts (EFs), embryonic stem cells with the disrupted IRAK gene were injected into C57BL/6J blastocysts and transferred to pseudopregnant mice. Embryos at day 15 of gestation were harvested. Fibroblast cell suspensions were prepared by trypsin treatment of the minced embryonic tissues. Fibroblasts derived from embryonic stem cells were enriched by culturing in DMEM with 10% FCS and 1 mg/ml G418. Fibroblasts after 3-4 wk of culture with G418 were used in these studies. Control EF cells with the wild-type IRAK gene were prepared from embryos of CD8-deficient mice (26). To prepare skin fibroblasts (SFs), mouse body skin was shaved, cut into small pieces, and then subjected to trypsin treatment. SFs in cell suspensions were cultured in DMEM with 10% FCS.
Cell Stimulation. Control and IRAK-deficient cells (9 ϫ 10 5 / plate) were plated overnight in 100-mm cell culture plates with DMEM containing 5% FCS. EF cells were kept in the presence of 200 g/ml G418. Before each experiment, cells were starved in serum-free DMEM for 4 h. The cells were then stimulated with IL-1 ␤ (R&D Systems, Minneapolis, MN) or TNF-␣ (Genzyme Corp., Cambridge, MA) at 37 Њ C. After stimulation, cells were scraped from the plates in ice-cold PBS and used immediately.
Western Blotting. Western blot analyses were carried out as previously described (27). For detection of IRAK and I B-␣ protein levels, NP-40 cell lysates (10 5 cells /sample) were separated on SDS-PAGE and transferred to nitrocellulose (MSI, Westboro, MA). The filters were immunoblotted with IRAK antiserum (1: 1,000 dilution) or rabbit antibody to I B-␣ (Santa Cruz Biotechnology). The bands corresponding to specific proteins were detected by horseradish-peroxidase-conjugated rabbit IgG and enhanced chemiluminescence (Amersham Corp.).
NF-B Mobility Shift Assay. After stimulation, the cells from 100-mm plates were suspended in 500 l of Buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 100 M EDTA, 100 M EGTA, 1 mM dithiothreitol (DTT), and 500 M PMSF) and incubated on ice for 15 min. After adding 30 l of 10% NP-40, the samples were centrifuged at 14,000 rpm at 4 Њ C for 30 s. The pellet was washed once with buffer A and resuspended in 50 l of buffer B (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) for 30 min at 4 Њ C. Nu-clear extracts were obtained by centrifugation of the samples at 14,000 rpm for 10 min. Protein concentrations were measured by the BCA method (Pierce, Rockford, IL). The consensus doublestranded oligonucleotide for NF-B binding (Santa Cruz Biotech) was labeled by T4 polynucleotide kinase. The nuclear binding reaction was carried out with 5 l of nuclear extract (10 g), 1 l of Poly(dI-dC), 1 l of labeled probe (100,000 cpm), 5 l 4 ϫ buffer (20 mM Hepes, pH 7.9, 20 mm MgCl 2 , and 1 mM EDTA) and 8 l of 25% glycerol for 30 min at room temperature. The binding complexes were separated on 6% polyacrylamide gel in 0.5ϫ Tris-buffered EDTA (Novex) and detected by autoradiography.
Northern Blot Analysis and IL-6 ELISA. For IL-6 mRNA detection, EF cells cultured in 100-mm plates were treated with IL-1␤ or TNF-␣ for 3 h at 37ЊC. Total RNA was extracted from EF cells with RNAzol (Tel-Test, Inc., Friendswood, TX). Northern blot analysis was performed as previously described (28)

Results and Discussion
IRAK-deficient Cells. To investigate whether IRAK is indispensable for IL-1 signaling pathway, IRAK-deficient mouse primary EF cells were prepared from mouse em- bryos injected with embryonic stem cells carrying a disrupted IRAK gene as described in Materials and Methods. The IRAK gene was disrupted by deletion of exons 5-7, which encode the NH 2 -terminal portion of the kinase domain, subdomains I-V, including conserved amino acids involved in ATP binding (Fig. 1 A). The deletion was confirmed by Southern hybridization (Fig. 1 B). Detection of only the disrupted IRAK allele in male embryonic stem cells suggests that the mouse IRAK gene is located on the X chromosome. Consistent with our finding, the human IRAK gene was also reported to be on the X chromosome (EMBL/GenBank'DDBJ accession number U52112). The absence of IRAK protein in EF cells containing the disrupted IRAK allele was demonstrated by immunoblotting with an anti-IRAK antiserum (Fig. 1 C). The lack of IRAK expression in IRAK-deficient EF cells enabled us to evaluate the importance of IRAK in IL-1 signaling.
Defective Activation of JNK and p38 in IRAK-deficient Cells. JNK and p38 are strongly activated by IL-1, TNF-␣, cell stress, and LPS (29)(30)(31)(32)(33). JNK phosphorylates the transactivation domain of c-Jun, which is involved in the activation of activator protein 1-dependent genes, including genes involved in inflammatory responses (29)(30)(31). p38 is also involved in activation of gene expression and protein synthesis during inflammation, including IL-1-induced IL-6 and prostaglandin synthesis (34). The proximal events responsible for IL-1-induced JNK and p38 activation are only partially understood. We therefore compared IL-1-induced activation of JNK and p38 in IRAK-deficient and control EF cells. JNK activity was measured by in vitro kinase assays of JNK immunoprecipitates, using c-Jun as substrate. Activation of JNK was reduced in IRAK-deficient cells by two-to threefold at all concentrations of IL-1 tested, although not completely eliminated (Fig. 2). This defect in JNK activation was IL-1 specific, since TNF-␣-induced JNK activity was comparable in control and IRAK-deficient cells. Activation of p38 was measured in p38 immunoprecipitates using MAP-KAPK-2 as a substrate. IL-1-induced activation of p38 was also reduced in IRAK-deficient cells compared with control cells, by three-to fivefold at all concentrations of IL-1 tested. In contrast, TNF-␣-induced p38 activity was similar in control and IRAK-deficient cells (Fig. 2). These results demonstrate that IL-1-induced JNK and p38 activities are both specifically reduced in IRAK-deficient cells, and implicate IRAK as the most proximal signaling component in the activation of the JNK and p38 pathways.
Defective Activation of NF-B. IL-1 and TNF-␣ are the two most efficient activators of the NF-B family of transcription factors (21,35). NF-B remains in an inactive state when sequestered by IB in the cytoplasm. Phosphorylation of IB by IB kinases leads to ubiquitination and subsequent degradation of IB by proteasomes (18)(19)(20)(21). Released NF-B is then translocated to the nucleus, where it binds to regulatory sites in NF-B-induced genes (21). IB levels in IRAK-deficient cells and control cells treated with IL-1 were determined by immunoblotting with an IB-␣-specific antibody. At concentrations of IL-1 ranging from 10 pg/ml to 1 ng/ml, IB degradation was significantly less in IRAK-deficient cells as compared with control cells (Fig. 3 A). However, at 10 ng/ml IL-1, IB-␣ was degraded almost completely in both IRAK-deficient and control cells. IL-1 induction of the NF-B pathway was further investigated by examining NF-B DNA-binding activity in nuclear extracts (Fig. 3 B). Significant reduction in NF-B activation was observed in IRAK-deficient Defective Induction of IL-6. IL-1 signaling triggers cellular responses through the induction of various inflammatory gene products (1). IL-6 is strongly induced by IL-1 via activation of transcription factors including NF-B and activator protein 1, which bind to the enhancer elements of the IL-6 gene promoter (6). IL-6 in turn plays a crucial role in inflammation by mediating acute phase reactions (36). To determine whether the observed defects in IL-1-activated signaling result in impaired cellular responses, IL-6 induction in response to IL-1 was measured. Levels of IL-6 mRNA induced by various concentrations of IL-1 were significantly lower in IRAK-deficient EF cells compared with control cells (Fig. 4 A). Similar decreases were also found in IL-6 secreted from IRAK-deficient cells compared with control cells (Fig. 4 B, top), and at various times after IL-1 treatment (data not shown). In three independent experiments, IL-6 induction by IL-1 was reduced by three-to sixfold. Thus, the reduced IL-1 signaling in IRAK-deficient cells results in decreased IL-6 production. To confirm the defects in IL-1 signaling and cellular response observed in IRAK-deficient EF cells, SF cells were obtained from mice deficient in IRAK expression, as described in Materials and Methods. Consistent with the observation in EF cells, IL-1-induced IL-6 production was significantly decreased in IRAK-deficient SF cells compared with SF cells prepared from wild-type mice (Fig. 4 B,  bottom). IL-1-induced JNK activation and IB degradation were also found to be defective in IRAK-deficient SF cells (data not shown).

Role of IRAK in Regulating
Multiple IL-1 Signaling Pathways. Taken together, our data demonstrate that IRAK plays an important role in activating multiple IL-1 signaling pathways that lead to the induction of IL-1-responsive gene products such as IL-6. In the absence of IRAK, induction of JNK and p38 activities was significantly reduced at all concentrations of IL-1 tested, although not completely eliminated. Our results suggest that IRAK is required for optimal induction of JNK and p38 kinases and that loss of its function cannot be completely compensated for by IRAK-2 or other related kinases. Activation of JNK and p38 have been reported to be mediated by cascades of upstream kinases including MAP-2, MAP-3, and MAP-4 kinases (29). It remains to be determined at which levels within these cascades IRAK may act to induce JNK and p38 activity.
In contrast to the effects on JNK and p38, defects in NF-B activation in IRAK-deficient cells could be overcome by high concentrations of IL-1. Our interpretation is that related kinases such as IRAK-2 may be able to fully activate the NF-B pathway under these conditions. However, induction of cellular response to IL-1, such as IL-6 production, is dramatically reduced in IRAK-deficient cells even at high IL-1 concentrations. This suggests that activation of NF-B alone is not sufficient for optimal induction of IL-1-mediated cellular responses. Induction of IL-6 and other IL-1-responsive gene products may be mediated synergistically by multiple pathways, including activation of NF-B, and JNK/p38-mediated pathways. In the case of IL-6 secretion, the dramatic decrease in its induction by IL-1 in IRAK-deficient cells, even in the presence of full activation of NF-B DNA-binding activity, may result from de- creased activation of JNK/p38-dependent signaling pathways. This possibility is supported by a recent report that p38 and other MAP kinases are involved in TNF-induced IL-6 gene expression via modulation of transactivation potential of NF-kB without affecting its DNA binding activity (37).
Our observation of significant reduction in multiple signaling pathways and in IL-6 production induced by IL-1 in IRAK-deficient cells suggests that inhibitors of IRAK or IRAK-related kinases may be therapeutically useful for the treatment of IL-1-mediated inflammatory diseases.
We thank G. Olini, Julie Culver, and Michelle Courtney for their excellent technical assistance. We also thank Jonathan Sprent, Avery August, Linda Joliffe, Lars Karlsson, and Michael Jackson for helpful discussions.