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J. Biol. Chem., Vol. 277, Issue 17, 14688-14694, April 26, 2002

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Phosphorylation Positively Regulates DNA Binding of the Carbon Catabolite Repressor Cre1 of Hypocrea jecorina (Trichoderma reesei)^*

Angela Cziferszky, Robert L. Mach^§, and Christian P. Kubicek

From the Section for Microbial Biochemistry and Gene Technology, Institute of Chemical Engineering, Technical University of Vienna, Getreidemarkt 9-166, A-1060 Vienna, Austria

Received for publication, January 23, 2002, and in revised form, February 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cre1 of the ascomycete Hypocrea jecorina is a Cys₂His₂ zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser²⁴¹ within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser²⁴¹. Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser²⁴¹ (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Carbon catabolite repression is a means by which cells manage priority use of easy and fast metabolizable carbon sources over more complex ones. In Saccharomyces cerevisiae, mutations in MIG1 or in respective binding sites in Mig1 regulated promoters partially relieve the yeast from carbon catabolite repression (1-4). To form an active repressor complex, Mig1 requires the co-repressors Ssn6 (Cyc8)-Tup1 (5-8). Mig1 function is regulated by glucose-dependent nuclear import/export, Mig1 being nuclear when glucose is present and being transported to the cytoplasm when glucose becomes limiting (9). Concomitantly, Mig1 has been shown to be phosphorylated under derepressing conditions (10, 11). Present genetic evidence suggests that both of these events are mediated by the Snf1 kinase complex: a mutation in MIG1 suppresses the snf1 mutant defects in SUC2 and GAL1 expression (3, 4), indicating that SNF1 negatively regulates repression by Mig1. Furthermore, Mig1 is permanently localized in the nucleus in snf1 mutants, even under glucose-limiting conditions (9). Results from the mutations of all four phosphorylation target sites present in Mig1 suggest that some of them are phosphorylated in vivo, but elimination of none of them reduced phosphorylation as completely as snf1 (11). This points to the possibility that other kinases, indirectly under Snf1 control, may also phosphorylate Mig1 (12).

Several filamentous fungi are efficient industrial producers of various enzymes and secondary metabolites frequently subject to regulation by carbon catabolite repression. Yet the molecular details of glucose repression are only at the beginning of being understood: genes homologous to MIG1 have been cloned and characterized from several fungi, i.e. Emericella nidulans (13), Aspergillus niger (14), A. oryzae (GenBank^TM accession number AAK 11189), A. aculeatus (GenBank^TM accession number BAA 75519), H. jecorina (15-17), Trichoderma harzianum (17), Metarhizium anisopliae (18), Botrytis cinerea (GenBank^TM accession number Y16625), Cochliobolus carbonum (GenBank^TM accession number AAG 29826), Neurospora crassa (GenBank^TM accession number AF055464), Acremonium chrysogenum (19), Gibberella fujikuroi (GenBank^TM accession number Y16626), Humicola grisea (20), and Sclerotinia sclerotiorum (21). It is intriguing that the respective gene products share a high degree of similarity with each other and with the Mig1 homologue from the fission yeast Schizosaccharomyces pombe (22) throughout several parts of the entire amino acid sequence, whereas a high degree of similarity with Mig1 is only apparent in the N-terminal DNA-binding zinc finger domain. As the Cre protein C-terminal part exhibits several features characteristic for transactivation domains (13, 15) one may speculate that the regulation of Cre-mediated repression is different from that exerted by Mig1 in S. cerevisiae. In support of this, Vautard et al. (21) reported that S. sclerotiorum cre1 cannot complement a Mig1 mutant of S. cerevisiae. However, using A. nidulans as heterologous host, they found that the nuclear location of Cre1 is dependent on glucose as is Mig1 in yeast (23). Furthermore, a S. sclerotiorum Cre1 mutated in a postulated AMPK/Snf1-kinase target serine and expressed in A. nidulans leads to an allyl alcohol-sensitive phenotype when grown on glucose (24). Although the latter findings were not accompanied by corresponding changes in Cre1 localization, the other data nevertheless suggest that the mechanisms leading to carbon catabolite repression in yeast and filamentous fungi are largely conserved.

Trichoderma reesei, the anamorph of the ascomycete H. jecorina (25), is an industrially important producer of cellulases and hemicellulases and Cre1-dependent carbon catabolite repression has been demonstrated for some of these genes (26-28). Using H. jecorina as a model for Cre1 regulation, we will show in this paper that Cre1 is indeed a phosphoprotein, but that both the kinase phosphorylating H. jecorina Cre1, as well as the effect of phosphorylation, are different from that known for S. cerevisiae.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fungal Strains and Culture Conditions-- H. jecorina (T. reesei) QM 9414 was used as wild-type strain throughout these studies. The mutant strain H. jecorina RUT C-30 (ATCC 56756), carrying a truncated and non-functional cre1 gene (17), was used as the recipient strain for hph-mediated transformation experiments. Conditions for growth were as described previously (15, 26).

Expression of Truncated and Mutated Versions of Cre1-- The pCre1 series of expression vectors was developed from pGEX-cre1 (15), a derivative of pGEX-4T1 (Amersham Bioscience, Uppsala, Sweden), containing a DNA fragment encoding amino acids 54-292 of Cre1. To create the truncated versions, pGEX-cre1 was digested with SacI/NotI or with NcoI/NotI, respectively. Plasmids were religated, using the oligonucleotide Cre1_link240 to generate pCre1-(54-240)_, and blunt ending the 5' protruding ends with Sequenase Version 2.0 (Amersham Bioscience, Little Chalfont, UK) to generate pCre1-(54-250). To insert the respective point mutations, pGEX-cre1 was digested with SacI/NcoI and religated (i) with a stuffer created by annealing oligonucleotides Cre1_ANDEf and Cre1_ANDEr, thus leading to pCre1-(S241A), (ii) with a stuffer made from annealing oligonucleotides Cre1_ENDEf and Cre1_ENDEr resulting in pCre1-(S241E), and (iii) with a stuffer consisting of Cre1_SNDVf and Cre1_SNDVr leading to pCre1-(E244V).

To fuse the identified kinase target SNDE to GST, pGEX-4T1 was digested with BamHI/NotI and religated with a stuffer formed by annealing the oligonucleotides SNDEf and SNDEr, thus generating pGEX_SNDE1 containing the fusion GST::HSNDEDD.

All described expression vectors, i.e. pGEX-cre1, pCre1-(54-292), pCre1-(54-250), pCre1-(54-240), pCre1-(S241A), pCre1-(S241E), pCre1-(E244V), and pGEX_SNDE1, were transformed into Escherichia coli BL21 (Amersham Bioscience). Expression, purification, and thrombin cleavage were carried out as described by the manufacturers.

Vector Constructions-- For construction of plasmid pACEC1, a derivative of pRLM_EX30 (30) containing an additional single BamHI site, a 2.8-kb XhoI-HindIII fragment comprising a full-length copy of the E. coli hph (hygromycin B phosphotransferase-encoding) structural gene flanked by the 5' region of the H. jecorina pki1 and the 3' region of the H. jecorina cbh2 gene was cloned into the plasmid pMTL23 previously digested with XhoI and HindIII.

Vectors pACEC31 and pACEC41 carrying full-length copies of the cre1 gene harboring the mutations S241A and E244V, respectively, and the hph transformation marker were constructed as follows: the respective mutations were first introduced by PCR into a 250-bp cre1 fragment, using CreS241A-f and CreE244V-f, respectively, as the respective forward primers (Table I), CreMutr as the reverse primer (Table I) and pACEC2, a pUC19 (New England Biolabs, Beverly, MA) derivative, which harbors a 5.5-kb HindIII-EcoRI fragment containing the entire cre1 coding region plus its flanking sequences (15), as a template. PCR amplifications were performed with Taq^TM polymerase (Promega, Madison, WI) and a Trio Thermocycler (Biometra, Göttingen, Germany) according to the instructions of the manufacturer, using an initial denaturation cycle of 5 min at 95 °C, followed by 35 cycles of 1 min denaturation at 95 °C, 1 min annealing at 62 °C and 30 s elongation at 74 °C. The amplicons were cloned into pGEM-T (Promega) and, after verification of the mutations by sequencing, used to replace the respective SacI-NcoI fragment of the wild-type cre1 gene as follows: first a 4.6-kb ClaI-HindIII cre1 fragment of pACEC2 was cloned into pUC19, the SacI site in the multiple cloning site removed by digestion with EcoRI and SmaI, filling in the protruding ends with Klenow polymerase (Promega) and finally religating to yield pACEC21. After digestion of pACEC21 with SacI and NcoI, corresponding SacI/NcoI fragments (250 bp) of the PCR-mutated cre1 genes were cloned into it to obtain pACEC3 and pACEC4. From these, 4.6-kb BamHI/HindIII fragments of the mutated cre1 genes were cloned into BamHI/HindIII-digested pACEC1 to yield pACEC31 and pACEC41.

Fungal Transformation-- H. jecorinaRUTC-30 was co-transformed with the plasmids pRLM_EX30 (30) and pACEC2 to yield strain CK11. Strain HphT was derived from transformation of H. jecorina RutC30 with plasmid pRLM_EX30. H. jecorina recombinant strains SA19 and EV7, carrying the Cre1 mutant alleles S241A and E244V were prepared by transforming H. jecorina RUTC-30 with pACEC31 and pACEC41, respectively. All transformations and transformant purifications were carried out following the Biolistic Bombardment method as described by Hazell et al. (31) using a Biolistic PDS-1000/He System (Bio-Rad, Hercules, CA).

Transformants were streaked twice on selective media, and then transferred onto malt extract agar plates (Merck, Darmstadt, Germany) for sporulation. Spore suspensions were plated on selective medium to obtain colonies derived from single spores for further analysis.

Southern Blot Analysis-- Fungal genomic DNA was isolated as described elsewhere (32) and Southern hybridization was carried out as described by Sambrook et al. (29).

Northern Hybridization-- Total cellular RNA was isolated as described by Chomczynski and Sacchi (33) and Northern hybridization carried out according to standard protocols (29).

Preparation of Cell-free Extracts and Electrophoretic Mobility Shift Assays (EMSA)¹-- Preparation of cell-free extracts from H. jecorina suitable for phosphorylation experiments and EMSA were carried out as described previously (34). DNA binding was achieved by incubating 100 µg (cell free extract) or 25 ng (recombinant Cre1) of protein with 5 ng of the respective labeled oligonucleotide. Synthetic, double-stranded oligonucleotides Cre1WT and Cre1MU were generated as described previously (26) (i) by annealing the respective oligonucleotides Cre1WTf and Cre1MUf with a complementary synthetic oligonucleotide Cre op for EMSA with recombinant Cre1 protein and (ii) by annealing the respective oligonucleotides Cre1WTfwd and Cre1MUfwd with the complementary synthetic oligonucleotide Cre1rev for EMSA with cell-free extracts. After annealing, double strands were filled in using Sequenase version 2.0; for labeling [-³²P]dCTP was added to the fill-in reaction.

Phosphorylation and Dephosphorylation of Cre1-- Dephosphorylation of Cre1 proteins, produced either by expression in E. coli or present in H. jecorina cell-free extracts, was performed by incubation of 25 ng of recombinant protein or 100 µg of cell-free extract for 15 min at 30 °C with 20 units of bovine intestinal mucose alkaline phosphatase (BIMAP; Sigma) in a reaction mixture of 15 µl containing 5 mM Tris-HCl, pH 8.5, 0.1 mM MgCl₂, and 0.01 mM ZnCl₂.

For phosphorylation of Cre1, 25 ng of the dephosphorylated recombinant protein was incubated in a total volume of 20 µl containing 50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 1 µM EDTA, 1% (w/v) Me₂SO, and 0.6 mM ATP. The reaction was started by the addition of 5 units of protein kinase from porcine heart (Sigma) exhibiting 3',5'-cyclic AMP-dependent phosphorylation activity. Incubation was carried out for 30 min at 30 °C. In selected cases, a total amount of 18.5 kBq of [-³²P]ATP (111 mBq/mol) replaced the 0.6 mM ATP in the above described assay. The mixture from the incubation was then subjected to SDS-PAGE (labeled probes), followed by autoradiography (35) or used for EMSA (unlabeled probes). For both phosphorylation and dephosphorylation assays, controls with heat-inactivated (10 min; 68 °C) enzymes were always included.

Phosphorylation of GST:HSNDEDD by Cell-free Extracts of H. jecorina-- To identify phosphorylation of Ser²⁴¹ in the GST:HSNDEDD fusion by cell-free extracts derived from glucose grown cultures, 5 µg of BIMAP-treated GST::HSNDEDD and 2 mg of cell-free extract of H. jecorina were incubated under the above described conditions for phosphorylation with a total of 18.5 kBq of [-³²P]ATP (111 mBq/mol) per assay. Incubation was carried out for 30 min at 30 °C. Following incubation, the reaction was stopped by the addition of 200 µl of pre-swollen glutathione-Sepharose suspension (Amersham Bioscience), followed by thorough mixing and spinning down in an Eppendorf centrifuge. After washing the pellet five times with 500 µl of 50 mM Tris-HCl, pH 8, the pellet was incubated with 2 units of thrombin (15 min, room temperature). Thereafter, released HSNDEDD was separated from glutathione-Sepharose by centrifugation, followed by three washing steps with 5 volumes of 50 mM Tris-HCl buffer, pH 8.0. The radioactivity was measured both in the supernatant and in the gluthatione-Sepharose pellets.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recombinant Cre1 Protein Is Phosphorylated at Ser²⁴¹ by E. coli and in Vitro-- An analysis of the aa sequence of Cre1/CreA proteins from several filamentous ascomycetes including Trichoderma (Hypocrea) revealed the presence of several target sites for protein kinases, such as protein kinase C, casein kinase II, and cAMP- or cGMP-dependent protein kinase, which were conserved among these fungi (Fig. 1), but not in S. cerevisiae. In contrast, no Snf1 kinase-like target motif (XRX₂SX₃ (36)), as present in Mig1, could be found. To investigate whether the H. jecorina Cre1 protein indeed is a phosphoprotein, and which of the putative phosphorylation sites may function as a target for phosphorylation in vitro, we expressed a cre1 fragment encoding amino acids 54-292, which bears all the mentioned phosphorylation consensus sites (Fig. 1), as a GST fusion protein in E. coli. As shown in Fig. 2, this fusion protein (Cre1-(54-292)) could indeed be phosphorylated, yet efficiently only, if previously treated with bovine intestinal mucose alkaline phosphatase (not pretreated samples, data not shown). This indicates that the recombinant Cre1 protein is, at least partially, phosphorylated in E. coli. The GST control alone was not phosphorylated.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Schematic drawing of the Cre1 protein fragments used in the in vitro experiments throughout this study. Amino acid sequences of Cre proteins from several multicellular ascomycetes (H. jecorina (T. reesei) (Hj), T. harzianum (Th), G. fujikuroi (Gf), M. anisopliae (Ma), A. chrysogenum (Ac), N. crassa (Nc), S. sclerotiorum (Ss), B. cinerea (Bc), E. nidulans (En), A. niger (An), A. aculeatus (Aa), C. carbonum (Cc), H. grisea (Hg), and A. oryzae (Ao)) were used to identify the conserved regions given as black boxes. Roman numbered gray boxes indicate the different identified putative phosphorylation sites; I, protein kinase C site; II, cAMP- or cGMP-dependent protein kinase site; III, casein kinase II site. Length of the complete and of all truncated Cre1 proteins and the positions of the respective point mutations are indicated.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   In vitro phosphorylation of recombinant Cre1 proteins. GST fusion proteins were expressed in E. coli, purified, cleaved by thrombin, and dephosphorylated as described under "Experimental Procedures." GST alone was used as a control in the phosphorylation experiments. All lanes were loaded with 6.25 ng (1.25 ng/µl) of recombinant protein from the phosphorylation reaction. Pre-stained Broad Range Marker Proteins (Amersham Bioscience, Uppsala, Sweden) were applied in an additional lane; Mr of the marker proteins are given in kDa.

To identify which of the detected phosphorylation target sequences are phosphorylated in vitro, C-terminal truncated versions (Cre1-(54-250) and Cre1-(54-240); Fig. 1) were constructed and overexpressed. Among these, only Cre1-(54-250) served as an acceptor for phosphorylation, whereas Cre1-(54-240) did not (Fig. 2), thus localizing the phosphorylated amino acid between amino acids 240 and 250 (Fig. 1), i.e. within the sequence HSNDEDDHYH. To prove that Ser²⁴¹ within this region serves as an acceptor for phosphorylation, we mutated this Ser²⁴¹ to an Ala (Cre1-(S241A)). The result from this experiment (Fig. 2) demonstrates that the mutation renders Cre1 inert for phosphorylation. Consequently, we deduce that Cre1 is phosphorylated at Ser²⁴¹.

In Vitro Phosphorylation/Dephosphorylation of Ser²⁴¹ in Cre1 Regulates Binding to Its Target Sequence-- To investigate whether the phosphorylation status of Cre1 has any effect on its DNA binding ability, we tested the binding of dephosphorylated recombinant Cre1-(54-292) to the oligonucleotide Cre1WT (bearing two Cre1-binding sites; cf. Table I) by EMSA (electrophoretic mobility shift assay) analysis (Fig. 3A). Cre1WT has previously been demonstrated to span the essential Cre1-binding sites of the xyn1 promoter of H. jecorina (26). Two specific complexes, presumably representing the binding of a single and of two Cre1 molecules to the target sequence were observed with Cre1-(54/292). Dephosphorylation reduced their binding almost completely (Fig. 3A). A control, incubated with heat-inactivated BIMAP, produced no inhibition of the DNA binding activity (Fig. 3A, lane 3), and rephosphorylation of the dephosphorylated protein with varying amounts of protein kinase from porcine heart and cold ATP led to a regain of DNA binding activity (Fig. 3B, lanes 2-6). Coherent data were obtained with the truncated Cre1-(54-250) and Cre1-(54-240) proteins, thus further supporting the hypothesis of a regulatory role of Ser²⁴¹ (Fig. 3A, lane 5-10). All these data are consistent with a positive regulation of DNA binding of Cre1 by phosphorylation. Interestingly, incubation with increasing amounts of protein kinase favored the formation of the higher molecular weight Cre1-DNA complex, suggesting that the phosphorylated form of Cre1-(54-292) binds as a dimer.

View larger version (61K): [in this window] [in a new window]   Fig. 3.   EMSA analysis of the ability of various recombinant Cre1 proteins to bind to the labeled synthetic double stranded oligonucleotide Cre1WT (5 ng/lane). A, Cre1 proteins used: Cre1-(54/292), Cre1-(54/240), and Cre1-(54/250) or with GST elution buffer only (free). stand. indicates untreated, BIMAP dephosphorylated Cre1 protein, and ia.BIMAP controls with heat-inactivated alkaline phosphatase. B, binding of Cre1-(54/292) protein to Cre1WT after rephosphorylation by different amounts of units (U) of protein kinase from porcine heart (Sigma). free indicated controls with GST elution buffer only.

As all these experiments had been carried out with a truncated version of Cre1, we further investigated whether the effect of phosphorylation would also be observed with the full-length native Cre1 protein. To this end, cell-free extracts from glucose-grown cultures of H. jecorina QM 9414 were dephosphorylated as described above, and tested in EMSA. To prove the specificity of the Cre1-DNA complex, we included (i) controls with oligonucleotide Cre1MU containing mutated, non-binding Cre1 sites (Table I), and (ii) experiments with cell-free extracts from the Cre1 mutant strain H. jecorina RUT C-30. These results (data not shown) confirmed that dephosphorylation completely destroys the specific DNA binding of Cre1 present in cell-free extracts of H. jecorina. From these data, we conclude that Cre1 requires phosphorylation to be capable of binding to its target sequence.

Cre1-(S241A) and Cre1-(S241E) Mutants Bind Their Target Sites Permanently-- From the results described hitherto, we expected that a mutation in Ser²⁴¹ would lead to a loss of DNA binding. Consequently, we mutated Ser²⁴¹ to an Ala, and tested the effect of this mutation on the ability of Cre1 to bind to its target sequence. However, in contrast to this expectation, a recombinant Cre1-(S241A) mutant protein showed equally strong DNA binding as the control (compare Figs. 3A and 4A), despite its inability to accept phosphorylation, and a similar finding was obtained with the truncated recombinant Cre1 fragment Cre1-(54/240) (Fig. 3A), which terminates before Ser²⁴¹. We interpret these findings in such a way that the presence of Ser²⁴¹ in Cre1 requires its phosphorylation for Cre1 being able to bind to DNA, whereas in the absence of Ser²⁴¹ Cre1 can do so even without phosphorylation. This result was further endorsed by the fact that also a Cre1-(S241E) mutant protein shows permanent DNA binding in vitro (Fig. 4A).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   A, effect of the S241A and S241E mutations in recombinant Cre1 on its binding to oligonucleotide Cre1WT. Conditions were essentially as described in legend to Fig. 3A. B, in vivo binding of Cre1-(S241A) of cell-free extract from strain SA19 to oligonucleotide Cre1WT. Cell-free extracts of strains HphT, CK11, and SA19 were used, free depicts EMSA with GST elution buffer only. Nonlabeled competitive oligonucleotides (comp. oligo) Cre1WT and Cre1MU were employed as indicated. C, Northern blot analysis of cbh1 expression from strains HphT, CK11, and SA19 after cultivation on glucose (Glu) for 24 h and lactose (Lac) for 36 h. Lanes contain 20 µg of total RNA; filters were hybridized with the H. jecorina cbh1 gene. Hybridization with the H. jecorina act1 gene and the ethidium bromide-stained agarose gel are given as controls.

Interestingly, the Cre1-(S241A) and Cre1-(S241E) mutant proteins only formed the lower molecular weight Cre1-DNA complex, indicating that the mutation of Ser to Ala and Glu, respectively, results in binding as a monomer only. In contrast, Cre1-(54-240) exhibits both DNA-protein complexes.

To demonstrate that the results described so far are not just in vitro artifacts, we transformed H. jecorina RUT C-30 with the cre1 gene harboring the S241A mutation to obtain strain SA19. H. jecorina RUT C-30, transformed solely with the hph cassette (strain H. jecorina HphT), and with the wild-type cre1 gene (strain H. jecorina CK11) were used as controls. Integration of just a single copy of the transformation cassette into the fungal genome was confirmed by Southern blot analysis. These strains were then precultivated on 1% glycerol followed by replacement into the same medium containing 1% glucose. Cell-free extracts were prepared and used for EMSA analysis. DNA binding to the synthetic oligonucleotide Cre1WT was observed by cell-free extracts from H. jecorina CK11 as well as SA19 bearing the Cre1-(S241A) mutation. H. jecorina HphT, the negative control, completely lacked the respective shift produced by the Cre1 protein (Fig. 4B). Competition experiments with an excess of nonlabeled Cre1WT and Cre1MU proved the Cre1-DNA complex genuine.

A H. jecorina Cre1-(S241A) Mutant Shows Permanent Carbon Catabolite Repression of cbh1 Gene Expression-- To test whether this binding of Cre1 also results in the corresponding phenotype in vivo, we used the cbh1 (cellobiohydrolase I-encoding) gene, whose expression is derepressed in a Cre1-non functional background (28), as a model system. Cbh1 transcript cannot be detected on glucose in H. jecorina CK11, but is clearly detectable in the Cre1-defective strain HphT. In H. jecorina SA19, however, no cbh1 transcript accumulation is apparent (Fig. 4C). Also, no cbh1 transcript accumulates in H. jecorina SA19 during growth on the derepressing carbon source lactose, whereas its accumulation is detected in H. jecorina CK11 (Fig. 4C). The abundance of the cre1 transcript was similar in strains CK11 and SA19, ruling out the possibility that the finding may be due to either poor transcription of cre1 or cre1 transcript instability in strain SA19 (data not shown). These data therefore provide evidence that the S241A mutation confers permanent carbon catabolite repression in vivo.

The Casein Kinase II Target Consensus Is Essential for Cre1 Phosphorylation and DNA Binding-- Since the aa sequence C-terminal of Ser²⁴¹ is consistent with the consensus for casein kinase II phosphorylation ((S/T)X₂(D/E) (37)), we also mutated Glu²⁴⁴ to Val (Cre1-(E244V)). This mutation, which deactivates the binding site of casein kinase II, nearly completely abolished the DNA binding ability in vivo (Fig. 5A). A similar result was observed for the recombinant Cre1-(E244V) protein in vitro and its DNA binding could not be recovered by phosphorylation (Fig. 5B). Furthermore, the protein kinase from porcine heart failed to phosphorylate Cre1-(E244V) (Fig. 5C). These data indicate that Glu²⁴⁴ (or an acid amino acid residue) is essential for the consensus sequence mediating phosphorylation-dependent Cre1-DNA complex formation. This observation makes the involvement of a casein kinase II-like protein kinase likely.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   A, binding of Cre1 from cell-free extracts of strains HphT, CK11, and EV7 to the synthetic oligonucleotide Cre1WT, free depicts EMSA with GST elution buffer only. B, effect of the E244V mutation in recombinant Cre1 on its binding to oligonucleotide Cre1WT (lanes 3-5). Lane 3 shows results from BIMAP-treated Cre1-(E244V), followed by re-phosphorylation with respective amounts of units (U) of protein kinase from porcine heart (lanes 4 and 5). Conditions were essentially as described in the legend to Fig. 3 (A and B). C, in vitro phosphorylation of recombinant Cre1 proteins. Conditions were essentially as described in legend to Fig. 2. D, phosphorylation of the GST::HSNDEDD fusion (GST::SNDE) by H. jecorina QM9414 cell-free extracts. GST alone was used as a control (GST); "+" and "" indicate where alkaline phosphatase treatment was carried out. Incorporated 32P was measured both in the supernatant (s) and in the glutathione-Sepharose pellet (p). Values are given as % of the radioactive background remaining in the pellet (set to 100%) and are means of five separate experiments; error bars indicate the corresponding standard deviation. E, Northern blot analysis of cbh1 expression from strains HphT, CK11, and EV7 after cultivation on glucose (glu) for 24 h and lactose (lac) for 36 h. Lanes contain 20 µg of total RNA, filters were hybridized with the H. jecorina cbh1 gene. Hybridization with the H. jecorina act1 gene and the ethidium bromide-stained agarose gel are given as controls.

To demonstrate that H. jecorina contains a casein kinase II-like enzyme activity, we fused a heptapeptide of the sequence HSDNEDD to GST and employed this fusion as a substrate for phosphorylation assays with cell-free extracts from a glucose-grown culture of H. jecorina. Results shown in Fig. 5D document that cell-free extracts of H. jecorina triggered the incorporation of ³²P specifically into this peptide and therefore contain a kinase capable of phosphorylating Ser within this consensus sequence.

Mutation of Cre1-(E244V) in the Casein Kinase Consensus Leads to Carbon Catabolite Derepression of cbh1 Gene Expression-- Since the Cre1-(E244V) mutation leads to a loss of Cre1-DNA binding, a corresponding H. jecorina mutant is supposed to be carbon catabolite derepressed. To test this assumption, we created the H. jecorina mutant strain EV7 by transformation of H. jecorina RUT C-30 with the respective Cre1-(E244V) mutant gene and tested the consequences of this on cbh1 gene expression. As can be seen in Fig. 5E, there was no regain of Cre1 function in strain EV7, thus indicating that a Cre1-(E244V) mutation is nonfunctional in vivo. Growth of strain EV7 on the carbon catabolite derepressing carbon source lactose led to cbh1 transcript levels similar to those on glucose (Fig. 5E), which is in accordance with this interpretation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results described in this paper confirm recent findings in S. sclerotiorum by Vautard-Mey and Fevre (24) that phosphorylation of Ser²⁴¹ (Ser²⁶⁶ in Sclerotium Cre1) is important for its ability to act as a carbon catabolite repressor and extend their study by showing that phosphorylation of H. jecorina Cre1 at Ser²⁴¹ is essential for binding to its target sequence. This is in contrast to S. cerevisiae Mig1 where phosphorylation has no effect on DNA binding (38).

However, while the amino acid, which acts as a target of this phosphorylation, is the same as in S. sclerotiorum, our findings in H. jecorina are notably different in several respects: expression of the S266A-mutated Cre1 of S. sclerotiorum in A. nidulans resulted in the expected carbon catabolite derepressed phenotype (24). In contrast, in H. jecorina a S241A mutation (which corresponds to the S266A mutation in S. sclerotiorum Cre1) did not result in a carbon catabolite derepressed phenotype in vivo. Similar findings were also obtained in vitro as the Cre1-(S241A) mutant protein was able to bind to its DNA target. Obviously, in H. jecorina Cre1 unphosphorylated Ser²⁴¹ interfers with its DNA binding. It is tempting to speculate that the hydrogen bonding ability of the hydroxyl group of Ser²⁴¹ is involved in this phenomenon, as it could interact with other amino acids near or within the DNA-binding domain, and thus create an inactive form of Cre1. In support of this hypothesis, a Cre1-(S241E) mutant protein, which mimics the negative charge introduced by phosphorylation, also showed DNA binding. An interaction with the N-terminal DNA binding, rather than with the C-terminal part of the protein, is suggested by the findings that Cre1-(54-250) still exhibits phosphorylation-dependent DNA binding. However, further discussions of the mechanism of activation of Cre1 by phosphorylation must await a structural and functional analysis of its various domains. Such information may eventually also help to explain why a mutation of Ser²⁶⁶-Ser²⁴¹ in Cre1 leads to the contrary phenotype in S. sclerotiorum/A. nidulans and H. jecorina, respectively.

Another difference to the results obtained with S. sclerotiorum/A. nidulans is the protein kinase possibly involved. (24) speculated that Ser²⁶⁶ is imbedded into a Snf1/AMP-kinase recognition motif. However, this kinase type requires a hydrophobic amino acid at +4 relative to S/T, which does neither occur in S. sclerotiorum nor in H. jecorina (SHEED and SNDED, respectively; cf. Fig. 1). Furthermore, the presented data have shown that a Cre1-(E244V) mutant protein (i) is not phosphorylated in vitro; (ii) does not bind to its target sequence; and (iii) leads to a carbon catabolite derepressed phenotype in vivo. As a mutation at this position should not affect phosphorylation by members of the Snf1/AMP kinase family, we consider a role of an Snf1p-homologue of H. jecorina very unlikely. In support of this, a C. carbonum snf1 mutant is not affected in carbon catabolite repression (39). Thus, a possible role of Snf1 in carbon catabolite respression in filamentous fungi is still unclear and warrants a more detailed study.

On the other hand, the aa sequence flanking Ser²⁴¹ (SNDEDD) perfectly matches the recognition motif of casein kinase II ((S/T)XX(D/E); E/D at +1/+2/+4 or +5 increasing the specificity; (36), and this consensus would also be in accordance with a loss of phosphorylation upon mutation of the acid amino acid residue at +3 relative to Ser/Thr. We also note that this consensus is conserved within all ascomyceteous Cre aa sequences (Fig. 1), but not in yeasts. Casein kinase II (37, 40-47), a heterotetrameric serine/threonine kinase, has been demonstrated to play an important role in the regulation of a large number of transcription factors, thereby affecting nuclear transport, DNA binding (both negatively and positively), transactivation, repression, as well as protein stability (37, 40-47). Genes encoding the two subunits of casein kinase II have not yet been cloned from any filamentous fungus, but from S. cerevisiae (48), and in part from S. pombe (49) and Y. lipolytica (50). Also, the N. crassa genome data base (www-genome.wi.mit.edu/annotation/fungi/neurospora/) lists a clone with high similarity to S. cerevisiae casein kinase -subunit (supercontig 1.14, 25800-27000), and two clones (r5h03a1, c4f02a1) encoding protein fragments with the same similarity are present in the A. nidulans EST data base (www.genome.ou.edu/fungal.html). Our findings of a kinase activity in cell-free extracts of H. jecorina, capable of phosphorylating an oligopeptide containing the casein kinase II consensus sequence, is therefore not unexpected and supports the presence of a casein kinase II homologue in H. jecorina which requires an acidic amino acid residue in +3. In view of the mutation analysis of Cre1-(E244V) in vivo we consider it therefore likely that this is the kinase involved in phosphorylation of Cre1. Phosphorylation-dependent binding of zinc finger-containing DNA-binding proteins to their target sequences has already been described for several examples from higher eukaryotes (42-45, 47, 51), yet it is the first time that this has been found for a C₂H₂-type zinc finger protein from a filamentous fungus. In some of these cases (42, 43, 51), phosphorylation also occurred by casein kinase II, and the phosphorylation target was within a regulatory domain. These findings are consistent with our present data, and suggest that casein kinase II-dependent phosphorylation of a domain not binding to DNA is not an unusual mechanism for the regulation of DNA-binding zinc finger proteins.

Probably the physiologically most important difference between H. jecorina and S. cerevisiae carbon catabolite repression is that phosphorylation of the respective regulator protein (Cre1 versus Mig1p) has different effects, as it is obligatory for Cre1-dependent carbon catabolite repression in H. jecorina whereas it relieves S. cerevisiae from Mig1-dependent carbon catabolite repression. This implies fundamental differences in the signaling of carbon catabolite repression in these two fungi. In S. cerevisiae, Mig1p phosphorylation by the Snf1-kinase regulates nuclear import (9). In S. sclerotiorum and A. nidulans, a switch in compartmentation such that CreA/Cre1 becomes nuclear under carbon catabolite repressing conditions has also been demonstrated (23), but none of the six serine protein kinase motifs in S. sclerotiorum Cre1 appeared to be essential for this glucose-induced shift in localization (24). It is reasonable to assume that this finding also applies to H. jecorina Cre1, and that CreA/Cre1 nuclear import is therefore regulated by other mechanisms, such as e.g. interactions with other proteins and protein turnover. In support of this, we have recently demonstrated that in A. nidulans, a shift from carbon catabolite repression to derepression results in a degradation of CreA (52). Also, the recent discovery of the A. nidulans CreB (carbon catabolite repression) protein as a ubiquitin-processing protease (53) adds further support for protein degradation as an important process in fungal carbon catabolite repression.

	ACKNOWLEDGEMENT

Help and discussions by Dr. Joseph Strauss during the early phase of this project are gratefully acknowledged.

	FOOTNOTES

^* This work was supported by Fonds zur Förderung Wissenschaftlicher Forschung Grants P-10792 MOB (to C. P. K.) and P 12 406-GEN.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to the article.

^§ To whom correspondence should be addressed. Tel.: 43-1-58801-17251; Fax: 43-1-581-62-66; E-mail: rmach@mail.zserv.tuwien.ac.at.

Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M200744200

	ABBREVIATIONS

The abbreviations used are: EMSA, electrophoretic mobility shift assay; BIMAP, bovine intestinal mucose alkaline phosphatase; GST, glutathione S-transferase; aa, amino acid(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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