Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3*

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.

Although sessile, plants can respond adaptively to various environmental stimuli by altering their growth and development. Phototropism, or directional growth of a given organ (e.g. seedling stem) in response to a change in incident light direction, represents one such response (1)(2)(3). Plants utilize a specific class of photoreceptors, the phototropins, to sense direc-tional blue light (BL) 2 cues that induce phototropic responses (4 -6). Two phototropins, phot1 (phototropin 1) and phot2, are present in the model plant Arabidopsis thaliana, and both are functional serine/threonine protein kinase photoreceptors (7,8). Although much is known about the structural means by which phototropins absorb BL and thus activate the kinase domain, at present, no native substrate other than the phototropins themselves has been identified (4,5). Moreover, although it is clear that perception of directional BL leads to phototropic curvature through differential accumulation of and response to the plant growth regulator auxin (9), the mechanism by which phototropin activation initiates signaling leading to this signal output remains elusive.
NPH3 is a novel 745-amino acid plasma membrane-associated protein and is a member of a large family of highly related plant-specific proteins that includes RPT2 (5,10,15). Three characteristics define members of the NPH3/RPT2 family of proteins: 1) an N-terminal BTB (Broad-Complex/Tramtrack/ Bric-a-brac) domain (19,20), 2) an NPH3 domain (Pfam accession number PF03000) in the middle of each protein, and 3) a C-terminal coiled coil in most members of the family (5). Although the functional role(s) of these domains are not fully understood at present, the coiled-coil region of NPH3 is known to be necessary and sufficient for interaction with phot1 (10), whereas the BTB domain has been shown recently to interact with CULLIN3 (CUL3) in a heterologous insect cell expression system. 3 Based on paradigms established in fungal and animal systems, in which BTB domain-containing proteins function as substrate adapters in CUL3-based ubiquitin-protein ligase (E3) (21)(22)(23)(24)(25), it has been proposed that NPH3 may function as a core component of a CUL3-based E3 complex that is necessary for phototropic signal progression (5). Although this hypothesis is currently under investigation, the physical and genetic interactions between phot1 and NPH3 suggest that, whatever the biochemical function of NPH3, it will somehow be regulated by phot1. Given that phot1 is a light-activated protein kinase (26), it seems reasonable to predict that such regulation of NPH3 function could occur through reversible phosphorylation.
NPH3 has been shown previously to exhibit different electrophoretic mobility states that are dependent upon in vivo irradiation conditions; in particular, BL specifically induces a state change to higher mobility relative to mock-irradiated samples (10). It was proposed that differing phosphorylation states of NPH3 could account for the observed alterations in electrophoretic mobility (10). However, no direct supporting evidence for this conclusion has been reported. In this study, we use a combination of pharmacological treatments and immunoblot assays to conclusively demonstrate that the higher mobility state of NPH3 does result from dephosphorylation and that this dephosphorylation is dark-reversible. In particular, we show that the BL-dependent increase in the electrophoretic mobility of NPH3 is sensitive to in vivo treatment with protein phosphatase (PPase) inhibitors and that in vitro treatment of samples never exposed to light with -PPase can phenocopy the effects of in vivo BL exposure. We further show that exposure of Arabidopsis seedlings to PPase inhibitors results in significantly reduced BL-dependent phototropism, consistent with the action of PPase(s) in phototropic signal transduction. Finally, we also provide evidence that the BL-dependent change in the electrophoretic mobility of NPH3 is dependent upon the phot1 photoreceptor and does not require functional phot2, the cryptochrome cry1 or cry2, or the phytochrome phyA or phyB. These results suggest that absorption of BL by phot1 leads to the activation of a PPase(s) that dephosphorylates an inactive phosphorylated form of NPH3 to allow further progression of phototropic signaling.

EXPERIMENTAL PROCEDURES
Plant Growth Conditions and Light Treatments-Seedlings of A. thaliana L. accession Columbia (Col-0) and various mutant genotypes in this background were used as sources of microsomal membranes and for the phototropic assays described in this study. The mutants used in this study have been described previously: phot1-5 (7), nph3-6 (10), and phot2-1 and phot1-5 phot2-1 (27). Seedlings were grown either on agar-based medium or in liquid culture (depending upon the experiment) as described below. First, seeds were surface-ster-ilized using 30% (v/v) commercial bleach (20 min) and then washed five times with sterile water. Unless noted otherwise, seeds were next placed either on filter paper mounted on 1% agar-solidified half-strength Murashige and Skoog medium (28) in Petri plates or in 15 ml of half-strength Murashige and Skoog liquid medium in 125-250-ml Erlenmeyer flasks. The seeds were then cold-treated for 3 days in darkness before treatment with red light for 1 h to induce uniform germination (29). The plates or flasks were next transferred to complete darkness, and seedlings were allowed to grow for 3 days prior to any treatment. For growth of seedlings in liquid medium, the flasks were shaken at 50 -60 rpm throughout their growth and subsequent treatments up to tissue collection. For BL treatments, seedlings growing on agar medium were either mock-irradiated or exposed to unilateral BL (29) of various intensities (0.01, 0.1, 1.0, and 10 mol m Ϫ2 s Ϫ1 ) for different time intervals (1,5,30, and 240 min), whereas seedlings grown in liquid medium were irradiated with BL (1.0 mol m Ϫ2 s Ϫ1 ) from underneath the flasks with constant shaking.
The influence of the PPase inhibitors on phototropism was assayed as follows. First, 3-day-old etiolated seedlings grown vertically on microscopic glass slides coated with half-strength Murashige and Skoog agar medium were completely immersed in half-strength Murashige and Skoog liquid medium containing a given inhibitor (or Me 2 SO as a control) for 2 h. Next, seedlings on the glass slides were exposed to unilateral BL (2.0 mol m Ϫ2 s Ϫ1 ) for 12 h. After collection of seedlings, hypocotyl lengths and phototropic curvatures were measured using Scion Image software (Scion Corp., Frederick, MD). Student's t tests were performed to compare the mean responses of seedlings under different treatments.
Preparation of Microsomal Membrane Proteins-Microsomal membranes were isolated from seedlings after BL or inhibitor treatments as described by Liscum and Briggs (16) with noted exceptions. All manipulations described below were performed under dim red light in a 4°C temperature-controlled room. Seedlings were ground in ice-cold homogenization buffer (25 mM MOPS-NaOH (pH 7.8), 250 mM sucrose, 0.1 mM MgCl 2 , 8 mM cysteine, 10 mM NaF, 5 mM ⑀-aminocaproic acid, 1 mM benzamidine, and Complete protease inhibitor mixture (Roche Applied Science)), followed by ultracentrifugation at 100,000 ϫ g for 1 h, 15 min to pellet microsomes. Microsomal membranes were then resuspended in resuspension buffer (5 mM potassium phosphate (pH 7.8), 250 mM sucrose, 4 mM KNO 3 , 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydro-chloride, 5 mM ⑀-aminocaproic acid, 1 mM benzamidine, and Complete protease inhibitor mixture) using a Dounce homogenizer. The resuspended microsomal membranes were aliquoted and stored in Ϫ70°C until used. The concentration of proteins in the microsomal membrane preparations was determined by the Bradford colorimetric method.
In Vitro Dephosphorylation Using -PPase-All steps of in vitro dephosphorylation reactions were performed under dim red light. Briefly, 20 g of total microsomal proteins from etiolated seedlings were mixed with 400 units of -PPase (New England Biolabs) in manufacturer-supplied buffer supplemented with 2 mM MnCl 2 , 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and 0.5% Triton X-100 and incubated on ice for 5 min and then at 30°C for 30 min. Reactions were terminated by the addition of 0.25 volumes of 4ϫ electrophoresis sample buffer (180 mM Tris-HCl (pH 6.8), 40% glycerol, 4% SDS. 0.04% bromphenol blue, and 200 mM dithiothreitol). The samples were then heated at 90°C for 5 min and subjected to SDS-PAGE as described below.
SDS-PAGE and Immunoblot Analysis-Total microsomal proteins (12 g/sample) were separated on 9% SDS-polyacrylamide gels (30) until the 72 kDa-specific band of the prestained molecular mass marker (Fermentas, Hanover, MD) reached the end of the gel. SDS-PAGE-separated proteins were then transferred to a nitrocellulose membrane by electroblotting in 192 mM glycine, 25 mM Tris, and 20% methanol.
For immunodetection, the membrane was first blocked using 5% (w/v) dry milk in 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.05% Tween 20 (Tris-buffered saline (TBS)/Tween) for 3 h. This was followed by incubation with primary antibody raised against the C-terminal region of NPH3 (1:5000 dilution) (10) for 2 h in TBS/Tween plus 1% dry milk. The membrane was then washed three times with TBS/Tween, followed by incubation with secondary antibody. The secondary antibody used in this study was alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega Corp., Madison, WI) diluted to 1:15,000 in TBS/Tween plus 1% dry milk. Finally, the membrane was washed four times with TBS/Tween (without dry milk), and NPH3-specific bands were detected by chemiluminescence using Immobilon AP substrate (Millipore Corp. Billerica, MA).

Rapid, Reversible, and Light-dependent Post-translational
Modification of NPH3-A previous study has shown that Arabidopsis NPH3 can exist in different modified states depending upon whether seedlings are grown and kept in darkness or exposed to light (10). In particular, NPH3 present in etiolated seedlings treated with BL exhibits increased mobility upon SDS-PAGE relative to NPH3 in seedlings kept in darkness or exposed to red light (10). In this study, we examined in detail several properties of this BL-dependent change in NPH3 mobility.
We first examined the fluence rate and time dependences of this response by performing immunoblot analyses on total microsomal proteins isolated from 3-day-old etiolated seedlings exposed to various fluence rates of BL for different periods of time. NPH3 exhibited a clear shift in electrophoretic mobility in membranes from seedlings exposed to very low (0.01 mol , and high (10 mol m Ϫ2 s Ϫ1 ) fluence rates of BL for as little as 5 min ( Fig. 1A and supplemental Fig. S1). In fact, we observed little, if any, fluence rate dependence for the formation of the higher mobility state of NPH3 (hereafter referred to as lightstate of NPH3 (NPH3 LS )) over the complete range of light intensities examined. Additional time course studies over a narrower range of irradiation times from 5 to 30 min indicated that the maximal levels of NPH3 LS accumulated sometime between 5 and 10 min after the start of BL exposure (Fig. 1B), although a shift in mobility could be detected after as little at 1 min of irradiation (Fig. 1, A and C).
It is interesting to note that, after 240 min of BL exposure, some portion of immunoreactive NPH3 appeared to exhibit slightly retarded mobility compared with that of NPH3 LS observed at 30 min (Fig. 1A). Such a change in mobility could result either from a reduction/reversal in whatever modification is causing the dark-state NPH3 (NPH3 DS )-to-NPH3 LS change or from a simple increase in protein abundance. To test the latter possibility, the time course experiment at 1.0 mol m Ϫ2 s Ϫ1 BL was repeated in the presence of cycloheximide to prevent de novo synthesis of NPH3. Because the mobility of NPH3 was indistinguishable between 30 and 240 min in cycloheximide-treated samples ( Fig. 1C and supplemental Fig. S1), we concluded that the "thicker" band observed at 240 min in the samples without cycloheximide (Fig. 1A) resulted from a de novo increase in NPH3 protein abundance rather than an alteration in post-translational modification.
We next investigated whether NPH3 DS could be recovered from NPH3 LS by placing in vivo BL-treated seedlings back into darkness. Etiolated seedlings were irradiated for 2 h with 1.0 mol m Ϫ2 s Ϫ1 BL and then returned to darkness for various times (Fig. 1D). Indeed, we did observe nearly complete recovery of NPH3 DS from NPH3 LS in the absence or presence of cycloheximide ( Fig. 1, E and F). The recovery of NPH3 DS from NPH3 LS in darkness was visible within 5 min independent of cycloheximide treatment. Interestingly, the recovery of NPH3 DS from NPH3 LS in darkness followed the same kinetics as that observed for the conversion of NPH3 DS to NPH3 LS in BL ( Fig. 1, compare B and F). Together, these results clearly illustrate that the observed modification of NPH3 is post-translational, dependent upon light, and reversible.
Upon long-term (e.g. 12-24 h) exposure to BL, phot1 is known to move from plasma membrane to cytoplasmic locations (31), likely endomembrane compartments (32). Such prolonged light treatment also renders phot1 less immunodetectable in immunoblot analyses (32). In contrast, NPH3 was detectable as NPH3 LS after prolonged exposure to BL for 24 h at 2.0 mol m Ϫ2 s Ϫ1 at levels comparable to those observed in dark-grown samples (data not shown). Moreover, treatment of seedlings for 12 h with BL followed by transfer to darkness for 12 h allowed reversion of NPH3 LS back to NPH3 DS , similar to what we observed upon short-term light/dark treatments (data not shown).
Dark and Light States of NPH3 Represent Different Phosphorylated Forms of the Protein-It has been proposed previously that the post-translational modification(s) leading to altered mobility of NPH3 upon SDS-PAGE might reflect differing phosphorylation states of the protein (10). We therefore examined the influences of PPase inhibitors on the state change of NPH3 in response to BL. If the conversion of NPH3 DS to NPH3 LS indeed reflects a dephosphorylation event, we should be able to prevent this conversion by pharmacologically blocking the activity of PPases ( Fig. 2A).
As shown in Fig. 2B, we found that OKA failed to block the BL-dependent conversion of NPH3 DS to NPH3 LS in 3-day-old etiolated seedlings at concentrations below 1 M, whereas at 1 M, this state change was almost entirely prevented. These results indicate that NPH3 in dark-grown seedlings (NPH3 DS ) exists as a phosphorylated species. Moreover, given that OKA is a cell-permeable toxin that preferentially inhibits PP2A (IC 50 ϭ 0.1-1.0 nM) compared with PP1 (IC 50 ϭ 10 -100 nM) (33,34) and has no effect on PP2C (35), these results also suggest that the conversion of NPH3 DS to NPH3 LS in response to BL treatment occurs through dephosphorylation, likely mediated by either PP1 or PP2A.
To further investigate the role of PP1 and PP2A in the dephosphorylation of NPH3 DS , we employed two additional selective PP1/PP2A inhibitors: CN and ET. Both of these are potent membrane-permeable inhibitors with higher specificity for PP2A (CN, IC 50 ϭ 40 nM; and ET, IC 50 ϭ 970 nM) than for PP1 (CN, IC 50 ϭ 473 nM; and ET, IC 50 ϭ 5 M) (36 -38). As shown in Fig. 2 (C and D), treatment of seedlings with either CN or ET at concentrations Ͼ1 M inhibited the BL-dependent conversion of NPH3 DS to NPH3 LS , with complete blockage of the state change occurring at Ն50 M. These pharmacological studies are consistent with the interpretation that the BL-dependent formation of NPH3 LS results from the dephosphorylation of NPH3 DS and that the dephosphorylation response is mediated by either PP1 or PP2A.
NPH3 LS Can Be Generated from NPH3 DS by Treatment with -PPase in the Absence of BL-Because PP1 and PP2A inhibitors were able to prevent the in vivo BL-dependent conversion of NPH3 DS to NPH3 LS , we next investigated whether the state change could take place in the absence of light by simply exposing microsomal membranes to an active PPase in vitro. -PPase was chosen for these assays because it represents a highly active enzyme that has been shown to hydrolyze the phosphate group from seryl, threonyl, tyrosyl, and histidyl residues (39). Fig. 2E clearly demonstrates that the conversion of NPH3 DS to NPH3 LS can occur in the absence of BL in vitro, provided that FIGURE 1. Light-regulated shift in the electrophoretic mobility of Arabidopsis NPH3. A, shown are the results of immunoblot analysis of NPH3 from total microsomal proteins isolated from 3-day-old etiolated wild-type Arabidopsis seedlings (Col-0) exposed to 0.01, 0.1, 1.0, and 10 mol m Ϫ2 s Ϫ1 BL for different lengths of time (0, 1, 5, 30, and 240 min) and then separated by SDS-PAGE. Total microsomal proteins were obtained from nph3-6-null mutant seedlings (negative control) exposed to BL at the indicated fluence rates for 240 min. Equal loading of proteins was visualized by silver staining, and a corresponding gel image is shown in supplemental Fig. S1A. B, 3-dayold etiolated wild-type Arabidopsis seedlings (Col-0) were exposed to 1.0 mol m Ϫ2 s Ϫ1 BL, and total microsomal proteins were extracted at the indicated times and analyzed by immunoblotting with an anti-NPH3 antibody (␣-NPH3). C, 3-day-old wild-type Arabidopsis seedlings (Col-0) grown in liquid culture were pretreated with cycloheximide (CHX; 200 M) for 1 h prior to treatment with BL (1.0 mol m Ϫ2 s Ϫ1 ). Total microsomal proteins were extracted at the indicated times after the start of BL treatment and analyzed by immunoblotting with an anti-NPH3 antibody. Equal loading of proteins was again visualized by silver staining, and the gel image is shown in supplemental Fig. S1B. D and E, the effects of dark adaptation on the mobility state of NPH3 are shown. D shows a scheme of the experimental protocol. E and F, 1 h prior to BL exposure, 3-day-old etiolated wild-type Arabidopsis seedlings (Col-0) grown in half-strength Murashige and Skoog liquid medium were treated either with cycloheximide (ϩCHX; 200 M) (F) or with an equal volume of Me 2 SO solvent (ϪCHX) (E). The seedlings were next exposed to BL (1.0 mol m Ϫ2 s Ϫ1 ) for 2 h and then transferred back into darkness. Total microsomal proteins were extracted at the indicated times after transfer to darkness. The dark controls (D) represent protein extracted from pretreated etiolated seedlings kept in darkness for 240 min. NPH3-specific bands were analyzed by immunoblotting using an anti-NPH3 antibody. Dashed lines in A-C depict the lowest mobility edge of NPH3 DS , whereas dashed lines in E and F depict the highest mobility edge of NPH3 LS . JULY 6, 2007 • VOLUME 282 • NUMBER 27 an active -PPase is present. Spontaneous dephosphorylation or degradation of NPH3 was not observed when total microsomal proteins were incubated with the buffer alone (Fig. 2E). Moreover, when sodium fluoride and sodium orthovanadate, common chemical inhibitors of PPases (40), were used in the assay, -PPase failed to dephosphorylate NPH3 (Fig. 2E). These results are consistent with the conclusion that the interconversion of NPH3 between NPH3 DS and NPH3 LS results from the dephosphorylation/phosphorylation of the protein.

Reversible Phosphorylation/Dephosphorylation of NPH3
PP1 and PP2A Inhibitors Perturb Phototropic Curvature-A previous study has shown that BL-dependent proton pumping and stomatal opening in Vicia guard cells, processes since shown to be dependent upon phototropins (14), are suppressed by PPase inhibitor treatment (41). We therefore investigated whether the phot1-dependent phototropic curvature of Arabidopsis seedlings could be abrogated by PPase treatment (Fig. 3, A-D).
Intriguingly, we found that preincubation of seedlings in 50 M CN, a concentration that completely abolished the dephosphorylation of NPH3 DS (Fig. 2C), also abolished hypocotyl phototropism (Fig. 3, A and B). At lower CN concentrations, the inhibitory effects on both responses were reduced similarly ( Figs. 2C and 3, A and B). The effects of ET were similar to those of CN in that concentrations inhibiting phototropism most effectively (Fig. 3C) were similar to those most efficient in inhibiting in vivo dephosphorylation of NPH3 DS (Fig. 2D). In contrast to the effects of CN and ET, a concentration of OKA that maximally inhibited the conversion of NPH3 LS to NPH3 DS (1 M) (Fig. 2B) only minimally affected BL-induced phototropism (Fig. 3D). It has been reported previously that exposure of OKA to UV light (300 -400 nm) results in its degradation (42). As our BL source contains a trace amount of UVA light (364 nm peak, comprising Ͻ2% of the total incident quanta), the tempered effects of OKA on phototropic curvature compared with those on the dephosphorylation of NPH3 DS observed here may reflect reduced levels of active OKA present during the longer BL exposures utilized in the former experiments (12 h versus Յ2 h). We have also observed that exposure to higher concentrations of OKA did not enhance the repressive effects of the inhibitor (data not shown), consistent with this conclusion. Although the aforementioned results suggest that the phosphorylation state of NPH3 is linked with phototropic responsiveness, one could argue that exposure of intact seedlings to the high concentrations of PPase inhibitors simply results in repression of basal metabolism such that hypocotyl cell elongation is reduced to such an extent that differential growth for phototropic curvature is also impaired. However, this appears not to be the case relative to the present experiments because hypocotyl elongation was not dramatically affected by exposure to PPase inhibitors (Fig. 3, E-G). For example, 10 M CN had a slightly significant effect on total hypocotyl elongation (Fig. 3E) and inhibited phototropism by ϳ50% (Fig. 3A), whereas exposure to 50 M CN completely inhibited hypocotyl phototropism (Fig. 3A), but did not significantly influence total elongation (Fig. 3, B and E). Collectively, the pharmacological results are consistent with a model in which the BL-dependent dephosphorylation of NPH3 DS and development of phototropism are linked and indicate that the former may be a prerequisite for the establishment of the latter.
BL-dependent Dephosphorylation of NPH3 DS Requires Functional phot1-Not only are NPH3 and phot1 localized in the same cellular compartment, but they also interact with each other physically (10 -12). With deference to this knowledge and to the fact that phot1 mediates phototropic responsiveness under both low and high fluence rate BL conditions (8) that are capable of stimulating NPH3 dephosphorylation (Fig. 1), we wished to determine the role of phot1 and other photoreceptors in the BL-dependent conversion of NPH3 DS to NPH3 LS . As shown in Fig. 4A, unilateral BL exposure stimulated normal NPH3 DS -to-NPH3 LS conversion in etiolated seedlings containing functional phot1 (Col-0 and phot2-1 lanes). However, only lower mobility NPH3, indistinguishable in mobility from NPH3 DS in mock-irradiated wild-type seedlings (Fig.  4A, lane 1), was observed in seedlings lacking phot1 (Fig. 4A, phot1-5  and phot1-5 phot2-1 lanes). The results presented in Fig. 4B demonstrate that NPH3 in seedlings lacking phot1 was in fact present as the phosphorylated form (NPH3 DS ) independent of in vivo BL exposure because in vitro -PPase treatment of microsomal membranes isolated from such seedlings resulted in the conversion of NPH3 to a higher mobility state (NPH3 LS ), as occurred with -PPase-treated wild-type microsomes (Fig. 2E). It thus appears FIGURE 3. Inhibitors of PP1/PP2A action can block BL induction of hypocotyl phototropism, but not overall hypocotyl elongation. A-D, 3-day-old wild-type Arabidopsis seedlings (Col-0) grown vertically on slides coated with half-strength Murashige and Skoog agar medium were pretreated with solvent (Me 2 SO or water) as a control or with PPase inhibitors (CN, ET, and OKA) at the indicated concentrations for 2 h in darkness, followed by exposure to unidirectional BL (2.0 mol m Ϫ2 s Ϫ1 ) for 12 h. Phototropic curvatures were then measured from digital images as described under "Experimental Procedures." A minimum of 45 seedlings from three replicate experiments were used to generate each mean value in A, C, and D. Error bars represent S.E. A, treatment with micromolar CN; B, representative image of seedlings treated with CN; C, treatment with micromolar ET; D, treatment with nanomolar OKA. ***, significant differences (p Ͻ 0.001) between untreated (control) and treated seedlings as determined by Student's t test. E-G, shown is the influence of the PPase inhibitors on overall hypocotyl elongation. Three-day-old etiolated Arabidopsis seedlings (Col-0) grown vertically on a microscope slide coated with half-strength Murashige and Skoog agar medium were pretreated with solvent (Me 2 SO or water), CN, ET, or OKA at the indicated concentrations for 2 h in darkness and then exposed to unidirectional BL (2.0 mol m Ϫ2 s Ϫ1 ) for 12 h. The seedlings were scanned digitally, and the length of the hypocotyl was measured using Scion Image software. A minimum of 33 seedlings from three replicate experiments were used to generate each mean value in E-G. Error bars represent S.E. E, treatment with micromolar CN; F, treatment with micromolar ET; G, treatment with nanomolar OKA. *, significant differences (0.05 Ͼ p Ͼ 0.02) between untreated control and treated seedlings as determined by Student's t test.
that phot1 is dispensable with respect to NPH3 phosphorylation, but is necessary for the BL-dependent dephosphorylation of NPH3 DS .
In addition to phot1, NPH3 has also been shown to physically interact with RPT2 (11) and PKS1 (12). To understand the role these NPH3-interacting proteins might play in the state change of NPH3 in response to BL, we examined the phosphorylation state of NPH3 in rpt2 and pks1 pks4 seedlings treated with BL at 2.0 mol m Ϫ2 s Ϫ1 for 2 h. As shown in Fig. 4C, the NPH3 DS -to-NPH3 LS transition occurred normally in both rpt2 and pks1 pks4 backgrounds, indicating that neither RPT2 nor PKS1 is essential for this phot1-dependent response. Because the BLabsorbing cryptochromes cry1 and cry2 and the predominantly red light-absorbing phytochromes phyA and phyB are known to function as secondary receptors in the modulation of phot1dependent phototropism (44 -47), we also examined the possibility that these photoreceptors could influence the phosphorylation state of NPH3. However, as shown in Fig. 4C, the BL-dependent conversion of NPH3 DS to NPH3 LS was completely unaffected in a phyA phyB cry1 cry2 quadruple mutant, indicating that the influence of cry1, cry2, phyA, and phyB on phot1-dependent phototropism does not occur through alterations in the phot1 regulation of NPH3 phosphorylation status. Taken together, the results presented in Fig. 4 demonstrate that the primary phototropic photoreceptor, phot1, is the only essential receptor regulating the phosphorylation state of NPH3.

DISCUSSION
Reversible protein phosphorylation is arguably one of the best understood and most common post-translational means of regulating cellular processes (48 -50). Quite often, the phosphorylation state of a given protein alone determines the "on/ off" state of a given signaling process (51)(52)(53)(54). In this regard, it is intriguing to note that the three major morphogenic photoreceptor types in plants (cryptochrome, phytochrome, and phototropin) exhibit protein kinase activity (4,(55)(56)(57)(58)(59)(60)(61) and that protein phosphorylation/ dephosphorylation events are associated with their signaling pathways (5,(62)(63)(64)(65)(66). In this study, we have demonstrated that the phot1-interacting protein NPH3 is a phosphoprotein in etiolated seedlings and that the BL-induced dephosphorylation of this protein may be necessary for phot1-dependent phototropic signaling.
To date, just one genetic locus that is apparently indispensable for BL-induced phototropism has been identified: NPH3 (10,11,(15)(16)(17). This, together with the knowledge that the NPH3 protein interacts physically with phot1 (10,11,12) and at least two other presumed signaling components, RPT2 (11) and PKS1 (12), makes NPH3 a likely target for regulation to modulate phototropic signal output. Previous analysis had shown that the electrophoretic mobility of NPH3 from etiolated seedlings irradiated in vivo with BL was enhanced compared with that of NPH3 from mock-irradiated siblings when resolved by SDS-PAGE, consistent with some type of post-translational modification, possibly phosphorylation (10). The results presented here clearly demonstrate that differing mobility states are in fact a result of different phosphorylation states of NPH3, the dark state (NPH3 DS ) being more phosphorylated than the light state (NPH3 LS ) ( Figs. 1 and 2).
Although we have yet to identify the specific residues exhibiting differences in phosphorylation state between NPH3 DS and NPH3 LS , it is likely that seryl/threonyl groups are targets because the BL-dependent dephosphorylation of NPH3 DS is sensitive to three different compounds (OKA, CN, and ET) (Fig.  2, B-D) that inhibit PPases with specificity for phosphoserine and phosphothreonine (67). The compounds tested here inhibit both type 1 (PP1) and type 2A (PP2A) PPases, with each inhibitor having ϳ5-10-fold higher specificity for PP2A than for PP1 (34,37,38). On the basis of the observed concentration dependences for the inhibitor effects on the BL-induced dephosphorylation of NPH3 DS (Fig. 2, B-D), we conclude that this process is likely mediated by PP1. The finding that exposure of seedlings to micromolar levels of CN and ET also inhibited phot1-dependent phototropism (Fig. 3, A-C) without dramatically impinging upon hypocotyl elongation (Fig. 3, E and F) is consistent with a model in which the PPase-dependent dephosphorylation of NPH3 DS is a necessary component of the phototropic response (Fig. 5).
The involvement of a PPase in phototropin signaling may not be that surprising when one considers that PPases appear to play a role in a variety of photoresponses. For example, PP2A, FIGURE 4. In planta BL-induced dephosphorylation of NPH3 DS requires phot1. A, wild-type Arabidopsis Col-0, phot1-5, phot2-1, and phot1-5 phot21 seedlings were grown in darkness for 3 days and then either mock-irradiated (0) or exposed to BL (0.1 or 2.0 mol m Ϫ2 s Ϫ1 ) for 2 h. Total microsomal proteins were extracted and separated by SDS-PAGE. NPH3 was detected by immunoblotting with an anti-NPH3 antibody (␣-NPH3). B, shown is the in vitro dephosphorylation of total microsomal protein samples from Col-0 and phot1-5 seedlings using -PPase. Phosphatase reactions on microsomal samples from mock-and BL-irradiated seedlings were terminated by the addition of SDS-PAGE loading buffer. Samples were then separated by SDS-PAGE and subjected to immunoblot analysis. NPH3 was detected with an anti-NPH3 antibody. C, etiolated 3-day-old Col-0, rpt2, pks1 pks4, and phyA phyB cry1 cry2 seedlings were mock-irradiated or exposed to BL (2.0 mol m Ϫ2 s Ϫ1 ) for 2 h, followed by total microsomal protein extraction, SDS-PAGE, and immunoblot analysis with an anti-NPH3 antibody. Dashed lines depict the lowest mobility edge of NPH3 DS .
PP2C, and PP5 have each been shown to interact with phytochrome in a spectral-dependent fashion to modulate subsets of red light-induced responses, including control of hypocotyl elongation and induction of flowering (62,63,68). The BL-dependent, cryptochrome-mediated control of hypocotyl and cotyledon growth also appears to genetically require the function of a PPase, in this case PP7 (69). Finally, there is also evidence that PP1 and/or PP2A is important in the regulation of light-induced gene expression necessary for chloroplast development and function (70,71).
Relative to the potential function of PP1 in phot1-dependent phototropic signaling, it is worth noting that PP1 has been associated previously with BL-induced, phot1-dependent proton pump activity and stomatal opening in V. faba (41,72). Although the protein(s) targeted by this latter PP1 has not been identified, it is unlikely to be NPH3 because nph3-null mutants retain normal BL-responsive stomatal function (11,18). Thus, it appears that PP1 represents a common element in what otherwise appear to be distinct phot1-dependent signaling pathways (4,5,65).
The finding that the BL-induced conversion of NPH3 DS to NPH3 LS does not occur in seedlings lacking phot1 (Fig. 4A) but can occur in phot1 mutant extracts treated with -PPase (Fig.  4B) is interesting for two reasons. First, the latter result indicates that NPH3 DS present in the phot1 mutant background in fact exists as a phosphorylated protein and that phot1 activity is not required for its phosphorylation in darkness. The observation that the immunoreactive NPH3 protein present in etiolated phot1 phot2 double mutants exhibits an electrophoretic mobility indistinguishable from that of NPH3 DS in the wild type and phot1 single mutants (Fig. 4A) suggests that phot2 is also dispensable with respect to the phosphorylation of NPH3 in darkness. These results imply that a protein kinase(s) other than a phototropin must also interact with NPH3 at some time to mediate phosphorylation of the NPH3 protein (Fig. 5). The observed rapid dark recovery of NPH3 DS from NPH3 LS in wildtype seedlings (Fig. 1, E and F) suggests that the protein kinase acting on NPH3 may in fact be "constitutively" present ( Fig. 5). Thus, the phosphorylation state of NPH3 at any given point in time is likely to reflect the balance between constitutive phosphorylation and BL-induced, phot1-dependent dephosphorylation, where the PPase activity is apparently dominant to the kinase activity (Fig. 5).
Second, the failure of NPH3 DS to become dephosphorylated in response to BL in phot1 mutant backgrounds indicates that phot1 is required, however, for the presumed PP1 to act on NPH3 DS (Fig. 5). On the basis of the observed electrophoretic mobilities upon one-dimensional SDS-PAGE, we estimate that the fluence threshold for the dephosphorylation of NPH3 DS is between 1 and 3 mol m Ϫ2 (Fig. 1A). Although this fluence threshold is at least an order of magnitude lower than that necessary for BL induction of bulk autophosphorylation of phot1 in etiolated Arabidopsis seedlings as estimated from SDS-PAGE mobility (73), partial autophosphorylation of phot1 does occur at lower fluences (e.g. 1 mol m Ϫ2 ) (supplemental Fig. S1C) (74). There is also similarity in the temporal requirements for BL-induced phot1 autophosphorylation and NPH3 DS dephosphorylation, as well as dark recovery of the light sensitivity of the two responses ( Fig. 1) (73). These overlapping photobiological properties of phot1 protein kinase activation and presumed PP1-dependent dephosphorylation of NPH3 DS suggest that activation of the protein kinase domain of phot1 may be a prerequisite for activation of the PPase (Fig. 5). This conclusion is certainly in harmony with the findings that NPH3 DS remains phosphorylated in phot1 mutant backgrounds (Fig. 4A) and that phot1 kinase activity is required for phototropic responsiveness (26). 4 But how does phot1 activation stimulate the PPase action on NPH3 DS ? It seems appropriate to consider this question within the context of two broad alternative regulatory means: direct versus indirect. Direct regulation would require photoactive phot1 to physically interact with a PPase to regulate its ability to 4 E. Liscum, unpublished data. The results presented here show that NPH3, a protein required for all documented phototropin-dependent phototropic responses in Arabidopsis, exists in two states depending upon the light condition: a phosphorylated form in darkness, NPH3 DS (upper), and a dephosphorylated form in BL, NPH3 LS (lower). It appears that NPH3 DS represents the phototropic signaling-incompetent state, whereas NPH3 LS is capable of transducing phot1-perceived directional BL cues leading to phototropic responses, likely through the action of a CUL3-based E3 complex of which NPH3 LS is part. NPH3 is apparently phosphorylated by a currently unidentified protein kinase, the action of which on NPH3 is in opposition to a BL-and phot1-activated PPase, most probably PP1. Although it has not yet been experimentally determined how phot1 regulates the PPase acting on NPH3 DS , it is hypothesized that it occurs via an influence on an allosteric regulator of the PPase. CC, coiled coil. RBX, real interesting new gene/U-box-protein.
dephosphorylate NPH3 DS . Although there is currently no evidence for an interaction between a PPase and phot1, such an interaction cannot be formally discounted. So how could an interaction between phot1 and a PPase modulate the activity of the latter? Given the protein activity of phot1, one can envision a mechanism by which a PPase is converted from an inactive to active state via direct phosphorylation by photoactive phot1. Although certainly not unheard of, PPases are not typically regulated through phosphorylation by a protein kinase (75)(76)(77)(78). Alternatively, phot1 could exhibit an allosteric inhibition on an associated PPase in darkness that is relieved in response to conformational changes in phot1 as a result of light absorption (79 -82). Although allosteric regulation of PPase activity is very common (83,84), the observation that NPH3 DS is not dephosphorylated in the phot1 mutant background (Fig. 4A) indicates that simple removal of phot1 is not sufficient for derepression of the PPase. Thus, if phot1 is functioning as a direct allosteric regulator of a PPase that acts on NPH3 DS , it must do so as both an inhibitor and an obligate facilitator of the PPase. Although we cannot ignore this possible mode of regulation, it seems unnecessarily complex.
As opposed to direct regulation, phot1 could influence the activity of the presumed PP1 that dephosphorylates NPH3 DS through an indirect means that does not require physical interaction between phot1 and the PPase, yet such indirect regulation would be expected to involve similar phot1-dependent mechanisms as described above for direct regulation, the major difference being that at least one additional factor would be required for the transfer of signal from phot1 to the PPase. Relative to an allosteric mode regulation, light-induced conformational changes in phot1 could alter the interaction with and activity of an allosteric regulator of a PPase to allow activation of the latter. phot1 would have just one required role in an indirect allosteric mode of regulation, viz. alteration of the allosteric regulator (either an inhibitor or activator). This is in stark contrast to a direct mode of regulation, where phot1 must act as both an inhibitor and activator. In the absence of a non-phot1 substrate for the phot1 protein kinase (4), we propose that phot1 regulates the activity of a PPase that targets NPH3 DS for dephosphorylation through an indirect influence on an allosteric regulator of the PPase (Fig. 5).
Perhaps the most intriguing question to arise from this study is not how phot1 mediates the dephosphorylation of NPH3 DS , but rather how the conversion of NPH3 DS to NPH3 LS promotes phototropic signaling and response. The genetic requirement for NPH3 in phototropic responsiveness is well known (10,11,(15)(16)(17)85), yet data presented here suggest that it is not just the mere presence of NPH3 that is required for signal progression, but that the proper post-translational state (NPH3 LS ) must exist (Fig. 3). To recap, NPH3 LS is generated through a BL-and phot1-dependent dephosphorylation of NPH3 DS (Figs. 1 and  4). So what could the dephosphorylation of NPH3 DS do to trigger phototropic signaling?
As introduced earlier, although novel and plant-specific, NPH3 is a BTB domain-containing protein (10,15,21) that has been proposed to function as a core component of a CUL3based E3 complex (5). It is intriguing to note that the activity of E3 ligases can be influenced by phosphorylation (86). For exam-ple, it is well documented that the phosphorylation state of many cell cycle control proteins determines their recognition and subsequent ubiquitination by E3 ligases (87,88). More directly, the phosphorylation state of E3 components themselves can determine ligase activity (43, 89 -92). It therefore seems plausible that the light/dark interconversion of NPH3 between its phosphorylated and dephosphorylated forms acts as a switch to regulate the activity of a CUL3-based E3 complex of which NPH3 is part (Fig. 5). The finding that the dephosphorylation of NPH3 DS appears to be correlated with phototropism (Fig. 3, A-D) suggests that such an E3 will contain NPH3 LS when its function promotes phototropic signaling (Fig. 5).
To conclude, the results presented here indicate that the phototropic signaling protein NPH3 exists as a phosphorylated protein in darkness and is rapidly dephosphorylated, likely by PP1, in response to phot1 photoactivation by BL. It appears that the dephosphorylated form of NPH3 (NPH3 LS ) is required for phototropic signal progression, possibly through alterations in the activity of a CUL3-based E3 containing NPH3 (Fig. 5). Future studies will be aimed at identifying the kinase and PPase involved in this regulatory process, the mechanism by which phot1 modulates the activity of that PPase, and the exact nature of the signal output from a NPH3-CUL3 protein complex.