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

2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês

10.1074/jbc.m702551200

ISSN

1083-351X

Autores

Ullas V. Pedmale, Emmanuel Liscum,

Tópico(s)

Plant Molecular Biology Research

Resumo

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. 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 (1Esmon C.A. Pedmale U.V. Liscum E. Int. J. Dev. Biol. 2005; 49: 665-674Crossref PubMed Scopus (92) Google Scholar, 2Liscum E. Somerville C.R. Meyerowitz E.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2002: 1-21Google Scholar, 3Iino M. Häder D.-P. Lebert M. Photomovement. Elsevier Science Publishers B.V., Amsterdam2001: 659-811Google Scholar). Plants utilize a specific class of photoreceptors, the phototropins, to sense directional blue light (BL) 2The abbreviations used are: BL, blue light; E3, ubiquitin-protein ligase; PPase or PP, protein phosphatase; OKA, okadaic acid; CN, cantharidin; ET, endothall; MOPS, 4-morpholinepropanesulfonic acid; TBS, Tris-buffered saline; NPH3LS, light-state NPH3; NPH3DS, dark-state NPH3. 2The abbreviations used are: BL, blue light; E3, ubiquitin-protein ligase; PPase or PP, protein phosphatase; OKA, okadaic acid; CN, cantharidin; ET, endothall; MOPS, 4-morpholinepropanesulfonic acid; TBS, Tris-buffered saline; NPH3LS, light-state NPH3; NPH3DS, dark-state NPH3. cues that induce phototropic responses (4Christie J.M. Annu. Rev. Plant Biol. 2007; 58: 21-45Crossref PubMed Scopus (627) Google Scholar, 5Celaya R.B. Liscum E. Photochem. Photobiol. 2005; 81: 73-80Crossref PubMed Scopus (37) Google Scholar, 6Kimura M. Kagawa T. Curr. Opin. Plant Biol. 2006; 9: 503-508Crossref PubMed Scopus (54) Google Scholar). Two phototropins, phot1 (phototropin 1) and phot2, are present in the model plant Arabidopsis thaliana, and both are functional serine/threonine protein kinase photoreceptors (7Huala E. Oeller P.W. Liscum E. Han I.S. Larsen E. Briggs W.R. Science. 1997; 278: 2120-2123Crossref PubMed Scopus (605) Google Scholar, 8Sakai T. Kagawa T. Kasahara M. Swartz T.E. Christie J.M. Briggs W.R. Wada M. Okada K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6969-6974Crossref PubMed Scopus (571) Google Scholar). 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 (4Christie J.M. Annu. Rev. Plant Biol. 2007; 58: 21-45Crossref PubMed Scopus (627) Google Scholar, 5Celaya R.B. Liscum E. Photochem. Photobiol. 2005; 81: 73-80Crossref PubMed Scopus (37) Google Scholar). 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 (9Esmon C.A. Tinsley A.G. Ljung K. Sandberg G. Hearne L.B. Liscum E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 236-241Crossref PubMed Scopus (171) Google Scholar), the mechanism by which phototropin activation initiates signaling leading to this signal output remains elusive. The identification of phototropin-interacting proteins represents a potentially powerful approach to elucidate how these photoreceptors transduce directional BL signals. Five phototropin-interacting proteins have been identified in recent years: NPH3 (non-phototropic hypocotyl 3) (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar), RPT2 (root phototropism 2) (11Inada S. Ohgishi M. Mayama T. Okada K. Sakai T. Plant Cell. 2004; 16: 887-896Crossref PubMed Scopus (166) Google Scholar), PKS1 (phytochrome kinase substrate 1) (12Lariguet P. Schepens I. Hodgson D. Pedmale U.V. Trevisan M. Kami C. de Carbonnel M. Alonso J.M. Ecker J.R. Liscum E. Fankhauser C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10134-10139Crossref PubMed Scopus (128) Google Scholar), Vicia faba phot1A-interacting protein (13Emi T. Kinoshita T. Sakamoto K. Mineyuki Y. Shimazaki K.-I. Plant Physiol. 2005; 138: 1615-1626Crossref PubMed Scopus (14) Google Scholar), and a 14-3-3 protein (14Kinoshita T. Emi T. Tominaga M. Sakamoto K. Shigenaga A. Doi M. Shimazaki K.-I. Plant Physiol. 2003; 133: 1453-1463Crossref PubMed Scopus (123) Google Scholar). Of these proteins, NPH3 represents the most likely candidate to yield insights into how phot1 transduces directional BL cues to induce phototropic responses because mutants lacking this protein are completely aphototropic under phot1-dependent conditions (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar, 11Inada S. Ohgishi M. Mayama T. Okada K. Sakai T. Plant Cell. 2004; 16: 887-896Crossref PubMed Scopus (166) Google Scholar, 15Sakai T. Wada T. Ishiguro S. Okada K. Plant Cell. 2000; 12: 225-236Crossref PubMed Scopus (200) Google Scholar, 16Liscum E. Briggs W.R. Plant Cell. 1995; 7: 473-485Crossref PubMed Scopus (355) Google Scholar, 17Liscum E. Briggs W.R. Plant Physiol. 1996; 112: 291-296Crossref PubMed Scopus (113) Google Scholar) while retaining other major phot1 functions such as chloroplast movements (11Inada S. Ohgishi M. Mayama T. Okada K. Sakai T. Plant Cell. 2004; 16: 887-896Crossref PubMed Scopus (166) Google Scholar) and stomatal aperture control (11Inada S. Ohgishi M. Mayama T. Okada K. Sakai T. Plant Cell. 2004; 16: 887-896Crossref PubMed Scopus (166) Google Scholar, 18Lasceve G. Leymarie J. Olney M.A. Liscum E. Christie J.M. Vavasseur A. Briggs W.R. Plant Physiol. 1999; 120: 605-614Crossref PubMed Scopus (105) Google Scholar). The remaining four proteins either are involved in other phot1-dependent processes (V. faba phot1A-interacting protein and the 14-3-3 protein) (13Emi T. Kinoshita T. Sakamoto K. Mineyuki Y. Shimazaki K.-I. Plant Physiol. 2005; 138: 1615-1626Crossref PubMed Scopus (14) Google Scholar, 14Kinoshita T. Emi T. Tominaga M. Sakamoto K. Shigenaga A. Doi M. Shimazaki K.-I. Plant Physiol. 2003; 133: 1453-1463Crossref PubMed Scopus (123) Google Scholar) or are not absolutely necessary for progression of phot1-dependent phototropic signaling (RPT2 and PKS1) (11Inada S. Ohgishi M. Mayama T. Okada K. Sakai T. Plant Cell. 2004; 16: 887-896Crossref PubMed Scopus (166) Google Scholar, 12Lariguet P. Schepens I. Hodgson D. Pedmale U.V. Trevisan M. Kami C. de Carbonnel M. Alonso J.M. Ecker J.R. Liscum E. Fankhauser C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10134-10139Crossref PubMed Scopus (128) Google Scholar). 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 (5Celaya R.B. Liscum E. Photochem. Photobiol. 2005; 81: 73-80Crossref PubMed Scopus (37) Google Scholar, 10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar, 15Sakai T. Wada T. Ishiguro S. Okada K. Plant Cell. 2000; 12: 225-236Crossref PubMed Scopus (200) Google Scholar). Three characteristics define members of the NPH3/RPT2 family of proteins: 1) an N-terminal BTB (Broad-Complex/Tramtrack/Bric-a-brac) domain (19Aravind L. Koonin E.V. J. Mol. Biol. 1999; 285: 1353-1361Crossref PubMed Scopus (130) Google Scholar, 20Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth & Differ. 1995; 6: 1193-1198PubMed Google Scholar), 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 (5Celaya R.B. Liscum E. Photochem. Photobiol. 2005; 81: 73-80Crossref PubMed Scopus (37) Google Scholar). 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 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar), whereas the BTB domain has been shown recently to interact with CULLIN3 (CUL3) in a heterologous insect cell expression system. 3N. Zheng and E. Liscum, unpublished data. 3N. Zheng and E. Liscum, unpublished data. 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) (21Stogios P.J. Downs G.S. Jauhal J.J. Nandra S.K. Prive G.G. Genome Biol. 2005; 6: R82Crossref PubMed Google Scholar, 22Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (374) Google Scholar, 23van den Heuvel S. Curr. Biol. 2004; 14: R59-R61Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 24Pintard L. Willems A. Peter M. EMBO J. 2004; 23: 1681-1687Crossref PubMed Scopus (294) Google Scholar, 25Krek W. Nat. Cell Biol. 2003; 5: 950-951Crossref PubMed Scopus (39) Google Scholar), 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 (5Celaya R.B. Liscum E. Photochem. Photobiol. 2005; 81: 73-80Crossref PubMed Scopus (37) Google Scholar). 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 (26Christie J.M. Swartz T.E. Bogomolni R.A. Briggs W.R. Plant J. 2002; 32: 205-219Crossref PubMed Scopus (242) Google Scholar), 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 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar). It was proposed that differing phosphorylation states of NPH3 could account for the observed alterations in electrophoretic mobility (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar). 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 λ-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. 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 (7Huala E. Oeller P.W. Liscum E. Han I.S. Larsen E. Briggs W.R. Science. 1997; 278: 2120-2123Crossref PubMed Scopus (605) Google Scholar), nph3-6 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar), and phot2-1 and phot1-5 phot2-1 (27Kagawa T. Sakai T. Suetsugu N. Oikawa K. Ishiguro S. Kato T. Tabata S. Okada K. Wada M. Science. 2001; 291: 2138-2141Crossref PubMed Scopus (527) Google Scholar). Seedlings were grown either on agar-based medium or in liquid culture (depending upon the experiment) as described below. First, seeds were surface-sterilized 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 (28Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (52758) Google Scholar) 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 (29Stowe-Evans E.L. Harper R.M. Motchoulski A.V. Liscum E. Plant Physiol. 1998; 118: 1265-1275Crossref PubMed Scopus (113) Google Scholar). 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 (29Stowe-Evans E.L. Harper R.M. Motchoulski A.V. Liscum E. Plant Physiol. 1998; 118: 1265-1275Crossref PubMed Scopus (113) Google Scholar) of various intensities (0.01, 0.1, 1.0, and 10 μmol m-2 s-1) for different time intervals (1, 5, 30, and 240 min), whereas seedlings grown in liquid medium were irradiated with BL (1.0 μmol m-2 s-1) from underneath the flasks with constant shaking. PPase Inhibitor Treatments—The following PPase inhibitors (Sigma) were used in this study: 9,10-deepithio-9,10-didehydroacanthifolicin (okadaic acid (OKA)); 2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-carboxylic acid anhydride (cantharidin (CN)); and a dicarboxylic acid derivative of CN, 7-oxybicyclo[2.2.1]heptane-2,3-dicarboxylic acid (endothall (ET)). OKA and CN were dissolved in Me2SO, whereas ET was dissolved in water. To test the influence of these compounds on the electrophoretic mobility of NPH3, a given compound was added to liquid cultures of 3-day-old etiolated (dark-grown) seedlings at the indicated concentrations with or without cycloheximide (Sigma) dissolved in 100% Me2SO. Controls were treated with an equal volume of Me2SO alone. 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 Me2SO as a control) for 2 h. Next, seedlings on the glass slides were exposed to unilateral BL (2.0 μmol m-2 s-1) 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 (16Liscum E. Briggs W.R. Plant Cell. 1995; 7: 473-485Crossref PubMed Scopus (355) Google Scholar) 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 MgCl2, 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 × 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 KNO3, 2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 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 -70 °C until used. The concentration of proteins in the microsomal membrane preparations was determined by the Bradford colorimetric method. In Vitro Dephosphorylation Using λ-PPase—All steps of in vitro dephosphorylation reactions were performed under dim red light. Briefly, 20 μg of total microsomal proteins from etiolated seedlings were mixed with 400 units of λ-PPase (New England Biolabs) in manufacturer-supplied buffer supplemented with 2 mm MnCl2, 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 4× 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 μg/sample) were separated on 9% SDS-polyacrylamide gels (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) 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) (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar) 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 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar). 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 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar). 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 μmol m-2 s-1), low (0.1 μmol m-2 s-1), intermediate (1 μmol m-2 s-1), and high (10 μmol m-2 s-1) 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 light-state of NPH3 (NPH3LS)) 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 NPH3LS 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 NPH3LS 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 (NPH3DS)-to-NPH3LS change or from a simple increase in protein abundance. To test the latter possibility, the time course experiment at 1.0 μmol m-2 s-1 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 NPH3DS could be recovered from NPH3LS by placing in vivo BL-treated seedlings back into darkness. Etiolated seedlings were irradiated for 2 h with 1.0 μmol m-2 s-1 BL and then returned to darkness for various times (Fig. 1D). Indeed, we did observe nearly complete recovery of NPH3DS from NPH3LS in the absence or presence of cycloheximide (Fig. 1, E and F). The recovery of NPH3DS from NPH3LS in darkness was visible within 5 min independent of cycloheximide treatment. Interestingly, the recovery of NPH3DS from NPH3LS in darkness followed the same kinetics as that observed for the conversion of NPH3DS to NPH3LS 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 (31Sakamoto K. Briggs W.R. Plant Cell. 2002; 14: 1723-1735Crossref PubMed Scopus (331) Google Scholar), likely endomembrane compartments (32Kong S.G. Suzuki T. Tamura K. Mochizuki N. Hara-Nishimura I. Nagatani A. Plant J. 2006; 45: 994-1005Crossref PubMed Scopus (119) Google Scholar). Such prolonged light treatment also renders phot1 less immunodetectable in immunoblot analyses (32Kong S.G. Suzuki T. Tamura K. Mochizuki N. Hara-Nishimura I. Nagatani A. Plant J. 2006; 45: 994-1005Crossref PubMed Scopus (119) Google Scholar). In contrast, NPH3 was detectable as NPH3LS after prolonged exposure to BL for 24 h at 2.0 μmol m-2 s-1 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 NPH3LS back to NPH3DS, 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 (10Motchoulski A. Liscum E. Science. 1999; 286: 961-964Crossref PubMed Scopus (248) Google Scholar). We therefore examined the influences of PPase inhibitors on the state change of NPH3 in response to BL. If the conversion of NPH3DS to NPH3LS 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 NPH3DS to NPH3LS in 3-day-old etiolated seedlings at concentrations below 1 μm, whereas at 1 μm, this state change was almost entirely prevented. These results indicate that NPH3 in dark-grown seedlings (NPH3DS) exists as a phosphorylated species. Moreover, given that OKA is a cell-permeable toxin that preferentially inhibits PP2A (IC50 = 0.1-1.0 nm) compared with PP1 (IC50 = 10-100 nm) (33Smith R.D. Walker J.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 101-125Crossref PubMed Scopus (193) Google Scholar, 34Bialojan C. Takai A. Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1493) Google Scholar) and has no effect on PP2C (35Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar), these results also suggest that the conversion of NPH3DS to NPH3LS 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 NPH3DS, 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, IC50 = 40 nm; and ET, IC50 = 970 nm) than for PP1 (CN, IC50 = 473 nm; and ET, IC50 = 5 μm) (36Deruere J. Jackson K. Garbers C. Soll D. DeLong A. Plant J. 1999; 20: 389-399Crossref PubMed Google Scholar, 37Erdodi F. Toth B. Hirano K. Hirano M. Hartshorne D.J. Gergely P. Am. J. Physiol. 1995; 269: C1176-C1184Crossref PubMed Google Scholar, 38Li Y.M. Mackintosh C. Casida J.E. Biochem. Pharmacol. 1993; 46: 1435-1443Crossref PubMed Scopus (128) Google Scholar). As shown in Fig. 2 (C and D), treatment of seedlings with either CN or ET at concentrations >1 μm inhibited the BL-dependent conversion of NPH3DS to NPH3LS, with complete blockage of the state change occurring at ≥50 μm. These pharmacological studies are consistent with the interpretation that the BL-dependent formation of NPH3LS results from the dephosphorylation of NPH3DS and that the dephosphorylation response is mediated by either PP1 or PP2A. NPH3LS Can Be Generated from NPH3DS by Treatment with λ-PPase in the Absence of BL—Because PP1 and PP2A inhibitors were able to prevent the in vivo BL-dependent conversion of NPH3DS to NPH3LS, 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. λ-PPase was chosen for these assays because it represents a highly active enzyme that has been shown to hyd

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