Blue light-regulated molecular switch of Ser͞Thr in phototropin

Daisuke Matsuoka and Satoru Tokutomi*

Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan

Communicated by Winslow R. Briggs, Carnegie Institution of Washington, Stanford, CA, July 27, 2005 (received for review February 27, 2005)

Phototropin is a blue light photoreceptor for tropic responses, relocation of , and stomata opening in . Pho- totropin has two chromophoric domains named light-oxygen- voltage-sensing (LOV) 1 and 2 in the N-terminal half, and a serine͞threonine (Ser͞Thr) protein kinase motif in the C-terminal half. Concerning the kinase activity of phototropin, only autophos- phorylation has been detected so far. However, we found that phototropin can phosphorylate a protein other than phototropin itself. Bacterially expressed Arabidopsis phototropin 2 kinase do- main (KD) with GST-tag showed a constitutive kinase activity on casein, a common in vitro substrate of Ser͞Thr protein kinase. By using this in vitro assay system, the roles of each LOV domain were studied. Addition of LOV2 to KD (GST-L2-KD) inhibits the kinase activity that is canceled by light. This light activation of kinase disappeared on introduction of a mutation blocking photochemical reaction in the LOV2 domain. Accordingly, LOV2 domain acts as a major light-regulated molecular switch of casein phosphorylation. Interestingly, isolated LOV2 from the KD still binds to the KD in a light-dependent manner and functions in similar ways, indicating the role of LOV2 domain as an inhibitor of the kinase activity in the substrate phosphorylation. LOV1, in contrast, contributes little to the photoactivation in GST-L1-L2-KD; however, it acts as an at- tenuator of the light activation of the kinase by LOV2. Fig. 1. Expression of Atphot2 recombinant polypeptides in Escherichia coli. (A) Schematic drawing of an Atphot2 domain structure and three expression photoregulation ͉ photoreceptor ͉ phosphorylation ͉ LOV domain constructs of N-terminal deletions, GST-KD (1–574 deletion), GST-L2-KD (1– 362 deletion), and GST-L1-L2-KD (1–116 deletion), respectively. KD, kinase ight in the wavelength region from UV to far-red is an domain; L1, LOV1; L2, LOV2. (B) SDS͞PAGE of the three recombinant polypep- Limportant stimulus for plants that precisely regulates devel- tides purified by glutathione-Sepharose. GST-KD (leftmost lane), GST-L2-KD opmental and cell motility processes. To sense the light condi- (second lane from the left), and GST-L1-L2-KD (third lane from the left) were tions including intensity, quality, and direction, plants have stained by Coomassie brilliant blue. Molecular masses (kDa) of the markers acquired three major photoreceptors (1): , crypto- (right lane) are indicated to the right of the gel. chrome, and phototropin. Phototropin (2, 3), originally identified as a photoreceptor for in the vicinity of the chromophore on formation of the protein– tropic responses (4), has also been found to mediate FMN adduct, whereas Fourier transform infrared studies have relocations (5–7), stomatal opening (8), leaf expansion (9), and shown secondary-structural changes in phy3-LOV2. Recently, an rapid inhibition of hypocotyl elongation (10). Most plants have NMR study has reported light-dependent structural changes in two isoforms of phototropin, named phot1 and phot2 (2). In a part of the linker region in oat phot1 (19), and a small-angle (At), phot1 and phot2 share tropic responses x-ray scattering study has proposed a relative movement of the and also one of the chloroplast relocation responses, photoac- linker region to the LOV2 domain in Arabidopsis phot1 (20). cumulation, depending on fluence rate of light (6). Photoavoid-

These changes are hypothesized to lead the activation of the ance response, on the other hand, is mediated by only phot2 (5). kinase domain upon adduct formation (21). In contrast, stomatal opening is regulated redundantly by both Phototropin was originally found as a protein phosphorylated phot1 and phot2 (8). rapidly on light-illumination in a membrane fraction. Since then, The phototropins are composed of 900–1,000 amino acid autophosphorylation of phototropin has been established both residues and two prosthetic flavin mononucleotide (FMN) mol- in vivo (22) and in vitro (4). The autophosphorylation sites reside ecules (2, 3) (Fig. 1A Top). The N-terminal half has a pair of between the LOV1 and LOV2 domains and also in the N- FMN-binding domains with Ϸ100 residues designated light- terminal region (23, 24). A rescue experiment with a pho- oxygen-voltage-sensing (LOV) 1 and 2 (11), and the C-terminal half forms a serine͞threonine (Ser͞Thr) kinase domain con- totropin-deficient Arabidopsis mutant has revealed that photo- nected to the LOV2 domain with a linker region (Fig. 1A).

Upon absorption of blue light, FMN in the two LOV domains Freely available online through the PNAS open access option. undergoes a unique cyclic photochemical reaction through for- Abbreviations: At, Arabidopsis thaliana; KD, kinase domain; LOV, light-oxygen-voltage- mation and breakage of a covalent bond (12) with a highly sensing; PAS, Per-Arnt-Sim. conserved cysteine found in all of the LOV domains of pho- *To whom correspondence should be sent at the present address: Department of Biological totropin-like proteins (13–15). Crystal structures of the Chlamy- Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, domonas phot-LOV1 (16) and Adiantum phytochrome 3 (phy3)- Osaka 599-8531, Japan. E-mail: [email protected]. LOV2 (17, 18) have revealed very small conformational changes © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506402102 PNAS ͉ September 13, 2005 ͉ vol. 102 ͉ no. 37 ͉ 13337–13342 Downloaded by guest on September 28, 2021 active LOV2 domains play a predominant role in activating (Amersham Pharmacia Biosciences), equilibrated and eluted autophosphorylation by light in both phot1 and phot2 (25). with 100 mM NaCl͞25 mM Tris⅐HCl͞1mMNa2EGTA (pH 7.8). These studies indicate a correlation between the autophosphor- Purity of the polypeptides was examined with Coomassie bril- ylation of phototropin and phototropic responses; however, its liant blue and anti-GST IgG staining of SDS͞PAGE gels. involvement in the signal transduction is still unclear. Further- more, no kinase activity of phototropin, except for autophos- In Vitro Phosphorylation Assay. Phosphorylation assay was per- phorylation, has been detected so far. formed under dim red light. KD-containing polypeptides were In the present study, we searched for substrates for the kinase incubated with casein (dephosphorylated for protein kinase activity of Atphot2 in common substrates of Ser͞Thr kinase and assay, Sigma) at 30°C in a kinase reaction mixture containing 30 found that casein can be a good substrate. This observation that mM Tris⅐HCl (pH 7.8), 100 mM NaCl, 1 mM Na2EGTA, 10% 32 phototropin can phosphorylate proteins other than phototropin glycerol, 10 mM MgCl2,20␮M ATP, and 37 kBq of [␥- P]ATP. itself is unique. Using the in vitro phosphorylation system, we The reaction was terminated by the addition of a concentrated investigated the role of LOV1 and LOV2 in regulation of the SDS͞PAGE sample buffer followed by boiling for 3 min. Then, kinase activity by light and found that LOV2 acts as a light- the samples were run on SDS͞PAGE, and molecular masses of regulated molecular switch of constitutively active kinase do- the bands were estimated by their positions revealed by Coo- main and that LOV1 attenuates the light sensitivity of the switch, massie brilliant blue staining. Phosphorylation of the bands was indicating distinct roles for the two LOV domains. visualized with imaging plates and a bioimaging analyzer (Fuji). The analyzer also quantified each band in the autoradiogram. Materials and Methods Light-dependent phosphorylation was measured with GST-L2- Construction of Expression Vectors. By using cDNA of Atphot2 as KD, GST-L1-L2-KD, and their photoinactive homologues and a template, required DNA fragments with appropriate restric- GST-KD in the presence of purified LOV polypeptides by giving tion sites were synthesized with PCR and oligonucleotide prim- a mock irradiation or irradiating with white light. Fluence- ers. Amplified DNA was isolated, digested, and cloned into a response of the light-induced kinase activity was studied with pGEX4T1 bacterial expression vector (Amersham Pharmacia GST-KD, GST-L2-KD, and GST-L1-L2-KD by irradiating with Biosciences) as a translational fusion to the glutathione S- white light supplied with a combination of a fluorescent tube and (GST). The following polypeptides, fused with GST a variable power supply for 10 min at different fluence rates. in the N terminus, were prepared: GST-KD (575–915), GST- Fluence rates of the light were measured with a spectroradiom- L2-KD (363–915), GST-L1-L2-KD (117–915), GST-L1 (117– eter LI-1800 (Li-Cor, Lincoln, NE). 265), and GST-L2 (363–500). In addition to these wild-type polypeptides, their non-adduct-forming homologues were GST Pull-Down Assay. GST or GST-KD was charged to glutathi- prepared by mutating the adduct-forming Cys to Ala. The one-Sepharose 4B resin according to the manufacturer’s proto- homologues are GST-L2(C426A)-KD [GST-L2(C͞A)-KD], col. The resin was incubated for1hat4°Cinbinding buffer GST-L1(C170A)-L2-KD [GST-L1(C͞A)-L2-KD], GST-L1- containing 25 mM Tris⅐HCl (pH 7.8), 100 mM NaCl, 1 mM L2(C426A)-KD [GST-L1-L2(C͞A)-KD], and GST-L1(C170A)- Na EGTA, and 10% glycerol with each LOV domain in the L2(C426A)-KD [GST-L1(C͞A)-L2(C͞A)-KD]. The kinase- 2 presence or absence of white light. The resin was collected by inactive mutant of GST-KD was prepared by replacement of an centrifugation and washed with the binding buffer three times aspartate residue essential to the kinase activity by asparagine under the same light conditions. The sample was separated on (26) in subdomain VII of the KD and designated as GST- ͞ KD(D720N). The amino acid substitutions were introduced by SDS PAGE and stained with Coomassie brilliant blue. using a Quick Change site-directed mutagenesis kit (Strat- Results agene), following the instructions, and verified by DNA sequenc- ing with a CEQ2000XL DNA analysis system (Beckman Expression of Atphot2 Polypeptides in E. coli. To characterize the Coulter). molecular mechanism of photoregulation of the kinase activity, we used three GST-fused N-terminal deletion polypeptides of Expression and Purification of Recombinant Proteins. The E. coli Atphot2, GST-KD, GST-L2-KD, and GST-L1-L2-KD expressed JM109 strain transformed by each expression plasmid was grown in E. coli (Fig. 1A). Unfortunately, preparation of full-length at 37°C in LB medium containing 50 ␮g⅐mlϪ1 ampicillin until Atphot2 has been unsuccessful, possibly because of in vivo degradation. Each GST-fusion protein, purified by glutathione- OD600 reached 0.3, and then incubated with 0.1 mM isopropyl ␤-D-thiogalactopyranoside for 20 h at 20°C in darkness. The Sepharose, showed an approximate molecular mass of 65, 89, or following purification procedures were carried out at 0–4°C 117 kDa (Fig. 1B), respectively. GST-L1-L2-KD and GST- under dim red light. Bacteria were collected by centrifugation L2-KD preparations have multiple components with smaller and suspended in the extraction buffer containing 50 mM molecular masses. They were degradation products in the C- Tris⅐HCl (pH 7.8), 100 mM NaCl, 1 mM EGTA, 10% glycerol, terminal regions because all were recognizable by GST antibody and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were (data not shown). Judging from their molecular masses, they lysed by sonication, and the supernatant was mixed with gluta- lacked KD, and their effects on the kinase assay could be thione-Sepharose 4B (Amersham Pharmacia Biosciences). The ignored. resin was washed with the extraction buffer. The GST-fusion proteins were eluted with 10 mM reduced glutathione in the Constitutive Kinase Activity of GST-KD. To see whether phototropin extraction buffer and stored at Ϫ80°C until use. For the purifi- can phosphorylate substrates other than phototropin itself, we cation of GST-free LOV1 and LOV2, bacteria were collected by tested several common substrates of Ser͞Thr kinase, and we centrifugation, washed with PBS, and resuspended in PBS found that casein with magnesium ion is the most effective to containing 1 mM PMSF. The cells were lysed by sonication, and monitor the GST-KD kinase activity. GST-KD phosphorylated the supernatant was mixed with glutathione-Sepharose 4B. After casein in a time-dependent manner, and this phosphorylation washing the resin with PBS, the LOV polypeptides were cleaved was saturated within 30 min (Fig. 2A). Autophosphorylation of from GST tag with thrombin, which leaves a linker of five amino the GST-KD was undetectable. Because neither GST itself nor acid residues, Gly-Ser-Pro-Glu-Phe, to the N terminus of each GST-KD(D720), a kinase-inactive mutant, phosphorylated ca- LOV construct. The dissociated polypeptides from the gel were sein (Fig. 2B), the observed kinase activity can be concluded to purified further by gel filtration on a Sephacryl S-100 HR column come from the constitutive kinase activity of KD in phot2.

13338 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506402102 Matsuoka and Tokutomi Downloaded by guest on September 28, 2021 Fig. 2. Constitutive protein kinase activity of Atphot2 GST-KD. (A) GST-KD Fig. 4. Effects of isolated LOV domains on protein kinase activity of GST-KD. and casein, marked by arrows, were incubated for indicated times in a kinase (A) Kinase assay of GST-KD was carried out in the absence (Ϫ) or presence of reaction buffer as described in Materials and Methods. The samples were purified LOV1 (LOV1) or LOV2 (LOV2) proteins as in Fig. 2. The samples were separated on SDS͞PAGE, stained with Coomassie brilliant blue (Right), and incubated for 10 min under white light (L) or mock irradiation (D). SDS͞PAGE then visualized by autoradiography (Left). (B) GST-KD(D720N), a kinase- gels were stained with Coomassie brilliant blue (Right) and then visualized by inactive mutant of GST-KD (two left-most lanes) or GST (two right-most lanes) autoradiography (Left). (B) GST pull-down assay on the interaction between were incubated with casein as described for A. GST-KD and each LOV domain. GST or GST-KD was charged to glutathione- Sepharose 4B resin and incubated with either LOV domain under white light (L) or mock irradiation (D). After washing of the resin, the sample was Photoregulation of Kinase Activity in Atphot2 Polypeptides. Using separated on SDS͞PAGE gel and stained with Coomassie brilliant blue. the above in vitro kinase assay system, we studied the roles of each LOV domain in regulation of kinase activity. In contrast to activity. GST-L1-L2-KD also showed a similar photoactivation GST-KD, GST-L2-KD did not phosphorylate casein in the dark of the kinase activity that was reduced significantly by introduc- (Fig. 3A Upper), suggesting the inhibitory role of LOV2 domain. tion of a Cys͞Ala mutation into the LOV2 [Fig. 3B, GST-L1- Light irradiation, however, induced phosphorylation of casein ͞ ͞ L2-KD and GST-L1-L2(C A)-KD]. In contrast, when Cys/Ala that was abolished by introduction of a Cys Ala mutation into mutation was introduced into LOV1, there was little effect on the LOV2 domain (Fig. 3A Lower) indicating the involvement of this photoactivation even after saturating light irradiation [Fig. photoreaction of LOV2 in this photoinduction of the kinase 3B, GST-L1(C͞A)-L2-KD]. When both LOV domains lost pho- toreactions in GST-L1(C͞A)-L2(C͞A)-KD, no kinase activity was detected [Fig. 3B, GST-L1(C͞A)-L2(C͞A)-KD], suggesting a minor contribution of LOV1 photoreaction in the kinase photoactivation. These results indicate that LOV2 acts as a major light-regulated switch of kinase activity.

Photoregulation of Kinase Activity by Isolated LOV2. To see the photoregulation mechanism of the kinase activity by LOV2 PLANT BIOLOGY domain, effects of isolated LOV domains on the kinase activity were studied (Fig. 4A). Presence of LOV1 domain did not reduce the kinase activity of GST-KD either in the dark or under light. LOV2 domain, in contrast, inhibited kinase activity in the dark that was canceled under light in ways similar to those of GST-L2-KD. This light-induced cancellation was not observed in the Cys͞Ala mutant of LOV2 domain, indicating that the photoreaction is requisite. Pull-down assay of GST-KD with isolated LOV2 domain clearly showed the association in the dark and dissociation under light of LOV2 domain to GST-KD (Fig. 4B Right, LOV2 WT). This dissociation was not observed in its Cys͞Ala mutant (Fig. 4B Right, LOV2 CA), indicating again the Fig. 3. Light-regulated protein kinase activity of GST-L2-KD (A) and GST-L1- L2-KD (B), including their Cys͞Ala (C͞A) -mutated homologues. As described involvement of the photoreaction. Furthermore, the assay indi- in the legend of Fig. 2, kinase activity was detected by phosphorylation of cates that the resides in KD but not GST (Fig. 4B casein by giving either white light (Light) or mock (Dark) irradiations, respec- Left, LOV2). LOV1 domain, unexpectedly, also binds to tively, for indicated times. GST-KD in a light-independent manner (Fig. 4B Right, LOV1);

Matsuoka and Tokutomi PNAS ͉ September 13, 2005 ͉ vol. 102 ͉ no. 37 ͉ 13339 Downloaded by guest on September 28, 2021 phototropin in E. coli for photochemical (11, 13, 27) or structural (17, 18) studies. Its active kinase domain, however, has never been obtained so far. Bacterially expressed oat phot1 kinase domain with a calmodulin-binding protein (CBP) tag was inactive (23). Recently, blue light-dependent autophosphorylation of Vicia faba pho- totropins was detected in situ in E. coli by far-Western blotting analysis (24), suggesting possibilities to prepare phototropin polypeptides with a kinase activity by an E. coli expression system. In the present study, we succeeded in preparing Atphot2 polypep- tides with kinase activity except for the full-length product. They were expressed in E. coli as GST N-terminal fusion proteins. The GST tag (26 kDa) is far bigger than the CBP tag (4 kDa) and known to form a dimer in solution (28) that may contribute to the proper production of kinase-active phototropins in E. coli. GST-KD phos- phorylated casein, a common in vitro substrate of Ser͞Thr protein kinase (Fig. 2). Phototropin can phosphorylate proteins other than phototropin itself, and this action raises possibilities that target proteins of phototropin kinase may exist in vivo and be involved in the light-signal transduction. Autophosphorylation was undetect- able in GST-KD, although the possibility that its autophosphory- lation has already been saturated in E. coli cells cannot be excluded. The others two polypeptides, GST-L2-KD and GST-L1-L2-KD, showed light-enhanced autophosphorylation. Light enhancement of GST-L1-L2-KD is much stronger than that of GST-L2-KD (data not shown). Both of the autophosphorylations were inhibited competitively with casein, in which competition in GST-L2-KD is much more obvious than that in GST-L1-L2-KD. These data are consistent with the reported sites of autophosphorylation between LOV1 and LOV2 domains of phototropin (23, 24).

Distinct Roles of LOV Domains in Regulation of Kinase Activity. In Fig. 5. Fluence response of photoactivation of the kinase in the four GST-L2-KD, LOV2 acted as an inhibitor of phototropin kinase N-terminus-deleted polypeptides. GST-KD, GST-L2-KD, GST-L1-L2-KD, or GST- in the dark, and light canceled the inhibition through the L1(C͞A)-L2-KD was incubated with casein in kinase reaction buffer for 10 min cysteine-FMN adduct formation (Fig. 3A). GST-L1-L2-KD also at different fluence rates. (A) Phosphorylation of casein was visualized as showed a similar photoactivation of the kinase, in which pho- described in Materials and Methods. Total fluences of irradiated light in the toreaction of LOV2 is essential (Fig. 3B). Furthermore, kinase wavelength region from 330 to 500 nm are indicated on the top. (B) Fluence- of GST-KD was inactivated in the presence of purified LOV2 response curves of the photoactivation of the kinase. Phosphorylation was domain but not LOV1 domain in the dark, and the inactivation quantified as described in Materials and Methods and normalized to 1 at the by wild-type LOV2 domain is relieved by light, as in GST-L2-KD, saturated levels. Each symbol represents the average phosphorylation levels ͞ of three independent experiments. Symbols on the left indicate the phos- though not in a Cys Ala mutant (Fig. 4A). Thus, all of the results phorylation levels in the dark. clearly show that LOV2 acts as a light-regulated molecular switch of phototropin kinase in the casein phosphorylation. The observed role of LOV2 in the photoregulation is consistent with however, this binding is to GST or possibly the resin because the report that LOV2 domain plays a major role in mediating LOV1 was pulled down by GST (Fig. 4B Left, LOV1). light-dependent autophosphorylation of phototropin and hypo- cotyl phototropism (25). LOV1, in contrast, shows no inhibition Role of LOV1 Domain. To clarify the role of LOV1 domain in of kinase activity in GST-KD even in the dark (Fig. 4A) and little regulation of the kinase activity, we compared fluence response contribution to the photoactivation of kinase in GST-L1-L2-KD of the photoactivation of the kinase in the Atpho2 polypeptides (Fig. 3B). It should be noted, however, that the contribution of lacking LOV1 or LOV1 and LOV2 domains (Fig. 5). In GST- LOV1 to the light activation of kinase cannot be totally excluded L2-KD, kinase activity depended on light intensity, and phos- because the introduction of Cys͞Ala mutation into the LOV1 phorylation was saturated at total fluence of 2,120 ␮mol⅐mϪ2. domain brought a minor additive inactivation of the kinase GST-L1-L2-KD also showed a similar fluence response to light activity (Fig. 3B). Furthermore, introduction of Cys͞Ala muta- irradiation; however, the fluence-response curve is shifted to the tion in the LOV1 domain flattened the fluence response curve, higher fluence region. Phosphorylation reached saturation at a and kinase activity is decreased by Ϸ18% at the total fluence of total fluence of 7,160 ␮mol⅐mϪ2, which is Ϸ3.5 times larger than 2,120 ␮mol⅐mϪ2. These data suggest a little contribution of that of GST-L2-KD. Introduction of Cys͞Ala mutation to the LOV1 photoreaction to the light-activation of kinase activity. LOV1 domain did not change the fluence at saturation. How- Although bacterially expressed LOV1 domains are reported to ever, the shape of the fluence response curve is somewhat show similar photoreactions (27) and the crystal structure (16) is different from that of the wild type; it is rather straight compared similar to that of LOV2 (17, 18), their roles are still obscure. with that of the wild type with a sigmoidal shape. Recently, LOV1 domain of oat phot1 was reported to be dimeric in solution, whereas LOV2 domain was monomeric (29), suggesting Discussion the role of LOV1 as a dimeric site. Present results supplied Construction of in Vitro Assay System of Phototropin Kinase. Pho- information regarding the role of LOV1 domain. Addition of totropin has been shown to be autophosphorylated in response to LOV1 shifted the fluence-response curve of the kinase photoacti- blue light irradiation in either soluble extracts from phototropin- vation in GST-L2-KD to the 3.5 times higher fluence region (Fig. expressing insect cells (4, 6) or membrane fractions of Arabidopsis 5). This shift suggests that LOV1 acts as an attenuator of the (22). There are many reports of expressing LOV domains of photoactivation, possibly because of a stereochemical blocking of

13340 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506402102 Matsuoka and Tokutomi Downloaded by guest on September 28, 2021 Fig. 6. Schematic illustration of the roles of LOV1 and LOV2 domains in light regulation of substrate phosphorylation by KD in Atphot2. P, phosphate; S, substrate; LL, low light conditions; HL, high light conditions. (A, B, and C) GST-KD, GST-L2-KD, and GST-L1-L2-KD, respectively, where GST is not indicated.

the interactions between LOV2 domain and KD independently of LOV2 on casein phosphorylation in darkness is concluded the photoreaction of LOV1. Thus, LOV1 and LOV2 domains through its direct binding to KD (Fig. 4B) without a linker region showed distinct roles in photoregulation of kinase activity in between LOV2 and KD. Recently, an intactness of J␣-helix in Atphot2 that are illustrated schematically in Fig. 6. KD expresses the linker region (19) was reported to be requisite in photoregu- kinase activity constitutively (Fig. 6A) and LOV2-KD shows light lation of Atphot1 autophosphorylation (33). Present results, in activation of kinase (Fig. 6B). LOV1-LOV2-KD does not exhibit contrast, show that a dissected LOV2 domain preserves regu- the activation because of attenuation by LOV1 under low light latory activity of the kinase by light. Our purified LOV2 domain conditions; however, the attenuation is overcome under high light has an amino acid sequence additional to the core of LOV2 in conditions. its C terminus; however, it did not contain the J␣-helix. The J␣-helix acts as a part of molecular switch that may assist LOV2 Interactions Between LOV Domain and Kinase Domain. Present re- domain to bind KD in the dark, and the assistance may be sults propose a photoregulation mechanism of phototropin abolished by the photoreaction of LOV2 domain. Our results kinase, in which LOV2 domain acts as a molecular inhibitor of suggest that the photoreversible binding capacity is in the LOV2 the kinase through direct binding between them (Fig. 4), and this core and does not necessarily require the aid of the hinge region, inhibition is abolished by light. Among the known Ser͞Thr which may be necessary for the proper interaction and photo- protein , eukaryotic Per-Arnt-Sim kinase (PASK) has the regulation of kinase activity in an intact phototropin molecule. ␣ mechanism most similar to that of phototropin (30). PASK has There is another possibility that the dispensability of J -helix two PAS (PAS-A and PAS-B) domains in the N-terminal region, may reflect different molecular mechanisms for the autophos- and the C-terminal half forms a Ser͞Thr kinase domain-like phorylation and the substrate phosphorylation of phototropin. phototropin. The kinase activity is inhibited by direct interac- tions with PAS-A. Furthermore, the activity of substrate phos- Concluding Remarks. Atphot2 has been proven to phosphorylate a phorylation is increased by autophosphorylation of PASK, sug- substrate other than the phototropin itself. This article proposes gesting that blue light-dependent autophosphorylation of the presence of novel substrate(s) involved in the signal trans- phototropin may act in a similar way. On the other hand, kinase duction, e.g., different substrates in each of photoaccumulation domains of phototropin are known to be homologous to those of and photoavoidance responses of chloroplasts. In Atphot2, two PKA, which has the same phosphorylation site specificity as that LOV domains function in different ways in the in vitro phos- of autophosphorylation in oat phot1 (23). An inactive form of phorylation. Major roles of LOV2 domain in the photoregula- PKA is known to be composed of two regulatory and two tion are shown also with the substrate phosphorylation. Atphot2 catalytic subunits, the former of which inhibit the kinase activity regulates phototropic response of hypocotyls only at high light of the latter (31). Binding of cAMP to the regulatory subunit intensity (6), which may be explained by the attenuation of the light sensitivity by LOV1 domain. The present in vitro phosphor-

leads to disassembly of the tetramer and release of active PLANT BIOLOGY catalytic subunits. We, therefore, examined whether PKA is ylation assay system is useful not only to study the photoregu- lation mechanisms but also to find possible in vivo substrates of inactivated by Atphot2 LOV2 domain in the dark as GST-KD Atphot2. Investigations along this line will supply important was. PKA activity, however, was not affected by Atphot2 LOV2 information to clarify the signal transduction pathways of phot2. domain (data not shown). Amino acid sequence alignments of KD between phototropin and PKA show that phototropin has We thank Prof. W. R. Briggs of Stanford University for critically reading additional sequences between subdomains VII and VIII. A the manuscript and Prof. M. Iino of Osaka City University for allowing docking site of the regulatory and catalytic subunits resides in us to use a spectroradiometer. This work was supported by Japanese this region (32). It seems that the interaction site of LOV2 to KD Ministry of Education, Culture, Sports, Science, and Technology Grant is located in this region of phototropin. The inhibitory effect of 13139205 (to S.T.).

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13342 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506402102 Matsuoka and Tokutomi Downloaded by guest on September 28, 2021