Phosphorylation of the Parsley bZIP Transcription Factor CPRF2 Is Regulated by Light
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29476
ISSN1083-351X
AutoresFrank Wellmer, Stefan Kircher, Alexander Rügner, Hanns Frohnmeyer, Eberhard Schäfer, Klaus Harter,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe analysis of the complex network of signal transduction chains has demonstrated the importance of transcription factor activities for the control of gene expression. To understand how transcription factor activities in plants are regulated in response to light, we analyzed the common plant regulatory factor 2 (CPRF2) from parsley (Petroselinum crispum L.) that interacts with promoter elements of light-regulated genes. Here, we demonstrate that CPRF2 is a phosphoprotein in vivo and that its phosphorylation state is rapidly increased in response to light. Phosphorylation in vitro as well as in vivooccurs primarily within the C-terminal half of the factor, and is caused by a cytosolic 40-kDa protein serine kinase. In contrast to other plant basic leucine-zipper motif factors, phosphorylation of CPRF2 does not alter its DNA binding activity. Therefore, we discuss alternative functions of the light-dependent phosphorylation of CPRF2 including the regulation of its nucleocytoplasmic partitioning. The analysis of the complex network of signal transduction chains has demonstrated the importance of transcription factor activities for the control of gene expression. To understand how transcription factor activities in plants are regulated in response to light, we analyzed the common plant regulatory factor 2 (CPRF2) from parsley (Petroselinum crispum L.) that interacts with promoter elements of light-regulated genes. Here, we demonstrate that CPRF2 is a phosphoprotein in vivo and that its phosphorylation state is rapidly increased in response to light. Phosphorylation in vitro as well as in vivooccurs primarily within the C-terminal half of the factor, and is caused by a cytosolic 40-kDa protein serine kinase. In contrast to other plant basic leucine-zipper motif factors, phosphorylation of CPRF2 does not alter its DNA binding activity. Therefore, we discuss alternative functions of the light-dependent phosphorylation of CPRF2 including the regulation of its nucleocytoplasmic partitioning. Light is probably the most variable environmental factor controlling plant development. To monitor light quality and quantity, plants have evolved at least three different photoreceptor systems: the red/far-red reversible phytochromes, the blue/UV-A, and the UV-B photoreceptors (1Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. Kluwer, Dordrecht, The Netherlands1994Crossref Google Scholar). The most well understood of these photoreceptors is the phytochrome system (2Whitelam G.C. Devlin P.F. Plant Cell Environ. 1997; 20: 752-758Crossref Scopus (184) Google Scholar, 3Furuya M. Schäfer E. Trends Plant Sci. 1996; 1: 301-307Abstract Full Text PDF Google Scholar). Besides the search for appropriate mutants, other approaches have been used to understand the signal transduction mechanisms mediated by photoreceptors. (i) Characterizing the photoreceptors themselves and searching for interacting proteins, (ii) unraveling the role of signal mediators like Ca2+, calmodulin, cGMP, and phosphorylation events (4Bowler C. Chua N-H. Plant Cell. 1994; 6: 1529-1541Crossref PubMed Scopus (211) Google Scholar), and (iii) analyzing DNA-binding proteins that interact with promoter elements of light-regulated genes. As shown, for example, forchalcone synthase or chlorophyll a/b-binding protein genes, promoter elements that mediate light responsiveness frequently contain the palindromic DNA motif ACGT, that, depending on the adjacent nucleotides, is part of the so-called G-box (CACGTG) or C-box (GACGTC) sequences (5Terzaghi W.B. Cashmore A.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 445-474Crossref Scopus (461) Google Scholar). However, G- and C-boxes are not only found in the promoters of light-regulated genes but also in promoters of genes that respond to other exogenic and endogenic stimuli such as stress, hormones, and cell cycle-related signals (6Meshi T. Iwabuchi M. Plant Cell Physiol. 1995; 36: 1405-1420PubMed Google Scholar). Transcription factors containing a basic leucine-zipper motif (bZIP), 1The abbreviations used are:bZIPbasic leucine-zipper motifCPRFcommon plant regulatory factorNi-NTAnickel nitrilotriacetic acidPAGEpolyacrylamide gel electrophoresisEMSAelectrophoretic mobility shift assayAPalkaline phosphatasePVDFpolyvinylidene difluoridep40protein kinase with an apparent molecular mass of 40 kDap50protein kinase with an apparent molecular mass of 50 kDa as, for example, the common plant regulatory factors (CPRFs) from parsley (7Weißhaar B. Armstrong G.A. Block A. da Costa e Silva O. Hahlbrock K. EMBO J. 1991; 10: 1777-1786Crossref PubMed Scopus (262) Google Scholar, 8Armstrong G.A. Weißhaar B. Hahlbrock K. Plant Cell. 1992; 4: 525-537Crossref PubMed Scopus (102) Google Scholar, 9Feldbrügge M. Sprenger M. Dinkelbach M. Yazaki K. Harter K. Weisshaar B. Plant Cell. 1994; 6: 1607-1621PubMed Google Scholar, 10Feldbrügge M. Hahlbrock K. Weisshaar B. Mol. Gen. Genet. 1996; 251: 619-627PubMed Google Scholar, 11Kircher S. Ledger S. Hayashi H. Weisshaar B. Schäfer E. Frohnmeyer H. Mol. Gen. Genet. 1998; 257: 595-605Crossref PubMed Scopus (56) Google Scholar) and G-box binding factors from Arabidopsis (12Schindler U. Menkens A.E. Beckmann H. Ecker J.R. Cashmore A.R. EMBO J. 1992; 11: 1261-1273Crossref PubMed Scopus (225) Google Scholar, 13Schindler U. Terzaghi W.B. Beckmann H. Kadesch T. Cashmore A.R. EMBO J. 1992; 11: 1275-1289Crossref PubMed Scopus (142) Google Scholar, 14Menkens A.E. Cashmore A.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2522-2526Crossref PubMed Google Scholar), were shown to bind to the G-box or the C-box, respectively, in vitro as well as in vivo and form specific homo- and heterodimers (7Weißhaar B. Armstrong G.A. Block A. da Costa e Silva O. Hahlbrock K. EMBO J. 1991; 10: 1777-1786Crossref PubMed Scopus (262) Google Scholar,8Armstrong G.A. Weißhaar B. Hahlbrock K. Plant Cell. 1992; 4: 525-537Crossref PubMed Scopus (102) Google Scholar, 12Schindler U. Menkens A.E. Beckmann H. Ecker J.R. Cashmore A.R. EMBO J. 1992; 11: 1261-1273Crossref PubMed Scopus (225) Google Scholar, 13Schindler U. Terzaghi W.B. Beckmann H. Kadesch T. Cashmore A.R. EMBO J. 1992; 11: 1275-1289Crossref PubMed Scopus (142) Google Scholar). Since CPRF and G-box binding factors proteins, which have molecular masses between 35 and 45 kDa, are encoded by multigene families (15Menkens A.E. Schindler U. Cashmore A.R. Trends Biochem. Sci. 1995; 20: 506-510Abstract Full Text PDF PubMed Scopus (326) Google Scholar), it is difficult to define which and how many members directly act as transcription factors regulating a certain inducible gene. basic leucine-zipper motif common plant regulatory factor nickel nitrilotriacetic acid polyacrylamide gel electrophoresis electrophoretic mobility shift assay alkaline phosphatase polyvinylidene difluoride protein kinase with an apparent molecular mass of 40 kDa protein kinase with an apparent molecular mass of 50 kDa The regulation of the activities of these factors in response to light is poorly understood. However, recent studies showed that the DNA binding activities of several factors of the bZIP-type are regulated by their phosphorylation state (16Klimczak L.J. Collinge M.A. Farini D. Giuliano G. Walker J.C. Cashmore A.R. Plant Cell. 1995; 7: 105-115PubMed Google Scholar, 17Dröge-Laser W. Kaiser A. Lindsay W.P. Halkier B.A. Loake G.J. Doerner P. Dixon R.A. Lamb C. EMBO J. 1997; 16: 726-738Crossref PubMed Scopus (135) Google Scholar, 18Ciceri P. Gianazza E. Lazzari B. Lippoli G. Genga A. Hoschek G. Schmidt R.J. Viotti A. Plant Cell. 1997; 9: 97-108PubMed Google Scholar). On the other hand, it was demonstrated that bZIP proteins exist in the cytosol of dark-cultivated parsley cells (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar). From these bZIPs it was shown to be CPRF2 that is localized in the cytoplasm of dark-cultivated cells and transferred to the nucleus upon irradiation (20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar). A detailed physiological analysis revealed that phytochrome photoreceptors induce the nuclear import of CPRF2 (20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar). Regulation of transcription factor activity, therefore, could be caused also by differences in the subcellular partitioning of the factors in dark- and light-grown cells. In this study we identified a 44-kDa protein in the cytosol of dark-cultivated evacuolated parsley protoplasts that in vitro is very rapidly phosphorylated in response to irradiation. In correlation with this observation we show that in vivothe phosphorylation of the 44-kDa bZIP factor CPRF2 is also rapidly enhanced by light. Since red light is most effective in inducing both phosphorylation events, we conclude that the phytochrome photoreceptor system is involved in these photoresponses. CPRF2 is phosphorylated by a cytosolic 40-kDa protein kinase at least one serine residue within its C terminus. As shown by size exclusion chromatography, CPRF2 and its kinase elute in a molecular mass range of about 300 kDa, indicating that both proteins are part of protein complexes. Since the phosphorylation of CPRF2 does not interfere with its DNA binding activity, we propose that light-induced changes in the phosphorylation state might be involved in the regulation of the nucleocytoplasmic distribution of CPRF2. Protoplasts were prepared under dim-green safety light (21Schäfer E. Albrecht H. Optische Strahlungsquellen. Lexika-Verlag, Grafenau, Germany1978: 249-266Google Scholar) from a dark-grown parsley cell culture 6 days after subcultivation. The protoplasts were evacuolated and cytosolic extracts were isolated as described previously (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar, 20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar, 22Harter K. Frohnmeyer H. Kircher S. Kunkel T. Mühlbauer S. Schäfer E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5038-5042Crossref PubMed Scopus (50) Google Scholar, 23Frohnmeyer H. Hahlbrock K. Schäfer E. Plant J. 1994; 5: 437-449Crossref PubMed Scopus (28) Google Scholar). The restriction fragment encoding full-length CPRF2 was subcloned into theBamHI site of the pQE70 vector (Qiagen) to produce a fusion protein with a C-terminal (His)6 tag. Transformation of the vectors in Escherichia coli, expression and purification of the proteins on nickel nitrilotriacetic acid (Ni-NTA)-agarose were performed under denaturating conditions as described in the manufacturer's protocol (Qiagen). The purified protein was refolded by removing urea by gel filtration through NAP 5 columns (Amersham Pharmacia Biotech) against 25 mm Tris/HCl, pH 7.8, 100 mm NaCl, and 1 mm dithiothreitol. The protein content of the eluate was determined using a method that is based on Coomassie Blue (24Sedmark J.J. Grossberg S.E. Anal. Biochem. 1977; 79: 544-552Crossref PubMed Scopus (2506) Google Scholar). 20 μg of freshly prepared cytosol kept on ice was supplied with 1 μl of [γ-32P]ATP solution (10 μCi, 5000 Ci/mmol in aqueous solution, Amersham Pharmacia Biotech), mixed once with a micropipette, and irradiated for 30 s on ice with a slide projector using appropriate filters (21Schäfer E. Albrecht H. Optische Strahlungsquellen. Lexika-Verlag, Grafenau, Germany1978: 249-266Google Scholar,22Harter K. Frohnmeyer H. Kircher S. Kunkel T. Mühlbauer S. Schäfer E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5038-5042Crossref PubMed Scopus (50) Google Scholar). Reactions were stopped by addition of boiling SDS sample buffer (65 mm Tris-HCl, pH 7.8, 4 m urea, 10 mm dithiothreitol, 5.0% (w/v) SDS, 0.05% (w/v) bromphenol blue) and denatured for 5 min at 95 °C before SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar) and transfer to polyvinylidene (PVDF) membrane (22Harter K. Frohnmeyer H. Kircher S. Kunkel T. Mühlbauer S. Schäfer E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5038-5042Crossref PubMed Scopus (50) Google Scholar). Detection of phosphotyrosine residues with a monoclonal antibody (clone 1G2) was performed according to the manufacturer's instructions (Roche Molecular Biochemicals). For each assay about 1.5 × 107 evacuolated protoplasts were suspended in 200 μl of a modified phosphate-free hemagglutinin medium (26Hahlbrock K. Planta. 1975; 124: 311-318Crossref PubMed Scopus (70) Google Scholar) containing 0.4 m sucrose (buffer P). 250 μCi of [32P]phosphate (10 mCi/ml in aqueous solution, Amersham Pharmacia Biotech) were added and the sample incubated for 5 min at room temperature. During incubation, samples were either irradiated with a slide projector using appropriate filters and mirrors to avoid warming effects or kept in darkness (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar, 21Schäfer E. Albrecht H. Optische Strahlungsquellen. Lexika-Verlag, Grafenau, Germany1978: 249-266Google Scholar, 22Harter K. Frohnmeyer H. Kircher S. Kunkel T. Mühlbauer S. Schäfer E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5038-5042Crossref PubMed Scopus (50) Google Scholar). After irradiation, evacuolated protoplasts were washed with 500 μl of buffer P and frozen in liquid nitrogen. After thawing, the cells were lysed in 250 μl of ice-cold buffer (50 mm Tris/HEPES, pH 7.5, 150 mm NaCl, 0.1% (v/v) Triton X-100, 1 mmbenzamidin, 5 mm ε-aminocaproic acid, 2 mmphenylmethylsulfonyl fluoride, 1 μg/μl antipain, 1 μg/μl leupeptin, 1 mm 4-nitrophenyl phosphate, 1 mmsodium fluoride, 1 mm sodium pyrophosphate), and the extracts were clarified by centrifugation. Then 5 μl either of CPRF2 antiserum or the corresponding pre-immunoserum were added and the sample incubated for 2 h on ice followed by addition of 15 μl of protein A-Sepharose (Amersham Pharmacia Biotech) and incubation for 1 h on ice. The protein A-Sepharose was washed 6 times with Tris-buffered saline/Tween (TBST: 50 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 0.05% (v/v) Tween 20) and subsequently boiled in SDS sample buffer. Precipitates were analyzed by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar) followed by autoradiography. Prior to size exclusion chromatography cytosolic extracts were centrifuged at 100,000 × g for 1 h at 4 °C. 1 ml of the supernatant (corresponding to 5 mg of protein) was fractionated through a Fractogel EMD BioSEC 600-16 column (Merck), with 20 mmNaH2PO4, pH 7.5, 300 mm NaCl, and 1 mm dithiothreitol at a flow rate of 1 ml/min. Individual fractions of 4 ml were concentrated and desalted in Centricon 10 tubes (Amicon) against 20 mm NaH2PO4, pH 7.5, to a final volume of 100 μl. For calibration of the column the following size markers (Sigma) were used: apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), and cytochrome c (12.4 kDa). 2 μg of recombinant CPRF2 were mixed in a total volume of 20 μl with 50 μg of cytosolic protein and 1/10 volume of 0.3 m Tris/HCl, pH 7.4, 50 mm MgCl2, 1.2 mmCaCl2. To test the gel filtration fractions for CPRF2 phosphorylation activity, the cytosolic protein was replaced by 10 μl of the desalted and concentrated fractions. 1 min after addition of 5 μCi of [γ-32P]ATP to the samples the reaction was stopped with 500 μl of 6 m guanidine hydrochloride, 0.1m NaH2PO4, 0.01 m Tris, pH 8.0. The (His)6-tagged proteins were isolated on Ni-NTA-agarose, eluted with 50 μl of 8 m urea, 0.1m NaH2PO4, 0.01 m Tris, pH 6.3, and 100 mm EDTA and tested for [32P]phosphate incorporation as described above. Silver staining of Ni-NTA purified CPRF2 was performed according to Ref. 27Heukeshoven J. Dernick R. Electrophoresis. 1985; 6: 103-112Crossref Scopus (1242) Google Scholar. For fragment analysis of in vivo phosphorylated CPRF2, an immunoprecipitate of 6 × 107 red light-irradiated evacuolated protoplasts was prepared as described above and cleaved with formic acid (28Van der Geer P. Luo K. Sefton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-58Google Scholar). For analyzing the in vitro phosphorylation, 20 μg of recombinant CPRF2 was subjected to the phosphorylation reaction and subsequently purified on Ni-NTA-agarose as described above. For the analysis of phosphopeptides, the purified recombinant CPRF2 was cleaved by formiate (28Van der Geer P. Luo K. Sefton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-58Google Scholar). The fragments obtained upon formiate cleavage ofin vitro and in vivo labeled CPRF2 were separated by SDS-PAGE (29Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10498) Google Scholar). Subsequently, the gels were dried and exposed to x-ray films. For the detection, fragments obtained from endogenous CPRF2 gels had to be exposed to a Kodak BioMax MS imaging film for 21 days. For phosphoamino acid analysis, recombinant CPRF2 was labeled as described above and analysis was performed according to Refs. 28Van der Geer P. Luo K. Sefton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-58Google Scholar and30Neufeld E. Goren J.G. Boland D. Anal. Biochem. 1989; 177: 138-143Crossref PubMed Scopus (70) Google Scholar. Recombinant CPRF2 (0.2 mg/ml) was polymerized into the resolving gel of a 12% (w/v) acrylamide gel (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar). 10 μl of the concentrated and desalted fractions from size exclusion chromatography and the unfractionated cytosol were boiled in SDS sample buffer and loaded onto the gel. After SDS-PAGE the gel was washed twice in buffer A (50 mm HEPES, pH 7.4, 5 mmβ-mercaptoethanol) containing 20% (v/v) isopropyl alcohol, and then re-equilibrated in buffer A for 1 h and subsequently incubated with 6 m guanidine hydrochloride in buffer A for 1 h. To renature the proteins the gel was washed extensively with ice-cold 0.05% (v/v) Tween 20 in buffer A (18 h). Then the gel was equilibrated in kinase assay buffer (10 mm MgCl2, 90 μm sodium vanadate in buffer A) for 30 min. The phosphorylation reaction was performed in 10 ml of kinase assay buffer with 30 μm ATP and 60 μCi of [γ-32P]ATP for 1 h at room temperature. Afterward the gel was washed in 10% (w/v) trichloroacetic acid, 1% (w/v) sodium pyrophosphate until unreacted radioactivity was removed, dried, and the signals detected by autoradiography. For the phosphorylation kinetics of recombinant CPRF2, 200 μl of the following mixture were prepared: 33 mm creatine phosphate, 0.04 unit/μl creatine kinase, 7 mm MgCl2, 7 mm ATP, and 3 μg/μl cytosolic protein. Creatine phosphate and creatine kinase (Sigma) were used to regenerate the ATP pool during the kinetics (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar). The mixture was divided. One-half was incubated on ice with 0.1 unit/μl apyrase (Sigma) to hydrolyze the ATP while the other half was kept on ice. After 30 min, 10-μl aliquots of the mixtures were removed, representing ATP-free and ATP-containing cytosolic extracts and used as controls of the endogenous DNA binding activity of the cytosol. To the remaining reaction mixtures (90 μl each) 0.01 μg/μl CPRF2 were added. For each time point of the kinetics, 10-μl aliquots were removed, mixed with 1 unit of apyrase to stop phosphorylation activities and frozen in liquid nitrogen. The samples of the phosphorylation reactions and the cytosolic fractions of the size exclusion chromatography were analyzed using electrophoretic mobility shift assay (EMSA). For this a DNA probe according to the monomeric G-box (5′-AATTCTCCCTTATTCCACGTGGCCATCCGG-3′) or the monomeric C-box (5′-AATTCTCCCTTATCTGACGTCAGCATCCGG-3′) was used (20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar, 31Schulze-Lefert P. Dangl J.L. Becker-André M. Hahlbrock K. Schulz W. EMBO J. 1989; 8: 651-656Crossref PubMed Scopus (199) Google Scholar, 32Schulze-Lefert P. Becker-André M. Schulz W. Hahlbrock K. Dangl J.L. Plant Cell. 1989; 1: 707-714PubMed Google Scholar, 33Block A. Dangl J.L. Hahlbrock K. Schulze-Lefert P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5387-5391Crossref PubMed Scopus (105) Google Scholar). Preparation of the radioactively labeled probes as well as experimental conditions for EMSA, electrophoretic mobility supershift assay, and treatments of evacuolated protoplasts extracts with alkaline phosphatase (AP) were described previously (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar, 20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar). In two recent reports (11Kircher S. Ledger S. Hayashi H. Weisshaar B. Schäfer E. Frohnmeyer H. Mol. Gen. Genet. 1998; 257: 595-605Crossref PubMed Scopus (56) Google Scholar, 20Kircher S. Wellmer F. Nick P. Rügner A. Schäfer E. Harter K. J. Cell Biol. 1999; 144: 201-211Crossref PubMed Scopus (91) Google Scholar) we characterized the expression and intracellular distribution of three members of the parsley CPRF transcription factor family. In our present work, we are studying the post-translational regulation of CPRF activities. Since transcription factor activities are frequently modulated by phosphorylation (34Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1119) Google Scholar), we initially tested in vitro whether a light-induced phosphorylation of a cytosolic polypeptide occurs in the molecular mass range of about 35–45 kDa that could represent a modification of one of the CPRF proteins. For this purpose, cytosol obtained from dark-cultivated evacuolated protoplasts was used. The removal of the toxic and proteolytically very active content of the vacuole from the protoplasts is necessary for obtaining functionally intact parsley protein extracts (19Harter K. Kircher S. Frohnmeyer H. Krenz M. Nagy F. Schäfer E. Plant Cell. 1994; 6: 545-559Crossref PubMed Scopus (126) Google Scholar, 22Harter K. Frohnmeyer H. Kircher S. Kunkel T. Mühlbauer S. Schäfer E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5038-5042Crossref PubMed Scopus (50) Google Scholar). The cytosolic extract was supplemented with [γ-32P]ATP and the reaction mixture divided in three aliquots. Two aliquots were irradiated for 30 s either with red or white light and one further kept in darkness. The reactions were stopped by addition of boiling SDS sample buffer. After SDS-PAGE and transfer onto PVDF membrane, the phosphorylation pattern of cytosolic proteins was analyzed by autoradiography. As shown in Fig.1 A, several proteins with different molecular sizes were phosphorylated in a light-dependent manner. Of particular interest was the phosphorylation of a protein with a molecular mass of approximately 44 kDa corresponding well with that of the factor CPRF2. This protein was most strongly labeled in response to red light irradiation indicating the involvement of phytochrome photoreceptors. Since tyrosine phosphorylation is discussed to be involved in phytochrome signaling leading to the activation of the G-box-containing chalcone synthase promoter (35Bowler C. Yamagata H. Neuhaus G. Chua N.-H. Genes Dev. 1994; 8: 2188-2202Crossref PubMed Scopus (163) Google Scholar), the same samples used above were tested with an antibody specific for phosphotyrosine. Although several polypeptides constitutively cross-reacted with the anti-phosphotyrosine antibody, no staining of those proteins was observed that were differentially labeled in response to irradiation (Fig. 1 B). These data indicate that the light-induced phosphorylation of all these proteins including the 44-kDa polypeptide occurs most likely at serine and/or threonine but not at tyrosine residues. The importance of phosphorylation for the regulation of transcription factor activities is well studied in yeast and animals (34Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1119) Google Scholar). Since the molecular mass of the observed phosphorylated cytosolic 44-kDa protein corresponds with that of CPRF2, we took into consideration that both proteins are identical. Therefore, we tested whether CPRF2 is a phosphoprotein in vivo and whether its phosphorylation state is changed in response to light treatment. Dark-cultivated evacuolated protoplasts were in vivo labeled with [32P]phosphate and irradiated for 5 min with light of different wavelengths or kept in darkness. After lysis of the evacuolated protoplasts, CPRF2 was isolated by immunoprecipitation and assayed for phosphorylation by autoradiography (Fig.2 A). Compared with the dark control (Fig. 2 A, lane 4), irradiation with either far-red, red, or white light enhanced the incorporation of [32P]phosphate into endogenous CPRF2 with red light being most effective (Fig. 2 A, lanes 1–3). No signal could be detected when the pre-immunoserum was used for immunoprecipitation (Fig. 2 A, lane 5). These data show that irradiation rapidly increases the phosphorylation state of CPRF2. To be able to further characterize the observed phosphorylation, an in vitro assay was established using recombinant CPRF2 and cytosolic extracts. As shown in Fig. 2 B, lane 1, recombinant CPRF2 was phosphorylated by cytosolic extracts leading to a strong signal after autoradiography. In contrast, no signals could be detected using cytosolic extracts without recombinant CPRF2 (Fig. 2 B, lane 2). To map the phosphorylation site of CPRF2 in vitro as well as in vivo, phosphorylated and purified recombinant as well as endogenous CPRF2 were used for formiate hydrolysis. Formiate treatment cleaves peptide bonds between aspartate and proline (28Van der Geer P. Luo K. Sefton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-58Google Scholar) and should result, in the case of CPRF2, in three peptides of 6.2 kDa (corresponding to amino acid (aa) 1–56), 14.5 kDa (corresponding to aa 57–195), and 23 kDa (corresponding to aa 196–401) (Fig. 3 A). After hydrolysis the fragments were separated by SDS-PAGE and the phosphorylation pattern was analyzed by autoradiography. Independently of the fact, whether CPRF2 was phosphorylated in vitro (Fig.3 B) or in vivo (Fig. 3 C), the major phosphorylated peptide had an apparent molecular mass of about 25 kDa. This suggests that, on the one hand, the predominately modified amino acid residues are very likely identical within the in vitroand in vivo labeled CPRF2 and, on the other hand, are localized within the C-terminal half of CPRF2. To identify the phosphorylated amino acid, a phosphoamino acid analysis of in vitro phosphorylated recombinant CPRF2 was performed. In agreement with the results shown in Fig. 1 B the phosphorylation of CPRF2 was confined exclusively to serine residues (Fig.3 D). The molecular properties of the CPRF2-phosphorylating serine kinase were further characterized by extending our in vitrophosphorylation approach. For this purpose, cytosol was prepared from dark-kept evacuolated protoplasts. Subsequently, the cytosol was irradiated with white light and separated by size exclusion chromatography. The obtained fractions were tested for CPRF2 phosphorylating activities by addition of recombinant CPRF2 and [γ-32P]ATP. As shown in Fig.4 A, labeling of CPRF2 was mainly found in fractions peaking around a molecular mass of approximately 300 kDa. A weak CPRF2 phosphorylating activity could also be detected in fractions around the molecular mass marker of 45 kDa (Fig. 4 A, lanes 8–10). These results could be interpreted that the CPRF2 phosphorylating kinase is either a very large protein or associated with other peptides in a multiprotein complex. To test these possibilities, we performed an in-gel assay. For this, recombinant CPRF2 was polymerized into the matrix of an SDS-PAGE gel. Complete cytosol and those cytosolic fractions showing CPRF2 phosphorylating activity were separated on this gel and, subsequently, the proteins were renatured within the gel matrix. Finally, the gel was incubated in a [γ-32P]ATP containing buffer, washed, and analyzed by autoradiography. As shown in Fig. 4 B, two kinase activities of different molecular mass could be detected in the cytosol. A 40 kDa activity (p40) that eluted in the high molecular mass range (with a maximum of approximately 300 kDa) and a 50 kDa activity (p50) that was found in a lower molecular mass range (with a maximum of approximately 60 kDa). In-gel kinase assays under identical methodical conditions but without matrix-immobilized CPRF2 did not yield any detectable signals (data not shown). This demonstrates that the CPRF2 phosphorylating activities derived from substrate phosphorylation and not from autophosphorylation activity. Since the main phosphorylation activity for CPRF2 (Fig. 4 A) was observed in the same high molecular weight fractions as p40 these data strongly suggest that p40 is the cytosolic CPRF2-specific serine kinase. Moreover, the appearance of p40 in a molecular mass range of about 300 kDa indicates that this kinase forms a complex with other proteins. On the other hand, those fractions containing high amounts of p50 show onl
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