Search

PCL6, PCL7(PAP1), and PHO80 belong to the PHO80 subfamily of PCLs (PHO85 cyclins), share high homology in protein sequences, and function with some similarity. YLR190w, the substrate of PCL7-PHO85, shares homology with YJL084c in a 140-amino-acid region. In addition, YJL084c was reported as a PCL6-binding protein. Here, it was found that there was association between YJL084c and PCL7, and their interaction was confirmed by co-immunoprecipitation assay and GST pull-down assay. The in vitro translational product of YJL084c could be phosphorylated by PCL7-PHO85 complex. Also, the GST fusion protein of the middle region expressed in E.coli could be phosphorylated, while the amino terminal or the carboxyl terminal could not. Interestingly, PCL6-PHO85 complex had the same characters; effect of phosphate condition on the phosphorylation was shown in both PCL6-PHO85 and PCL7-PHO85. YPH499: Yjl084c :: LEU2 was constructed by homologous recombination. PUT4 was reported as a YJL084c-binding protein, but no difference was observed between wild strain and the Yjl084c null mutant on MP medium. In addition, the interaction between PHO81 and all of the three cyclins was analyzed.

A yeast two-hybrid screening by using PAP1 was performed to identify targets for PAP1-PHO85 cyclin-CDK complex. N-terminal fragment of protein YLR190w, a yeast gene encoding a 491 amino acids peptide, was identified, and its coding region was amplified by PCR. The interaction of PAP1 and YLR190w was confirmed by both two-hybrid assay and GST pull-down assay in vitro. The PAP1-PHO85 kinase complex obtained from the immunoprecipitates could phosphorylate GST-YLR190w expressed in E.coli, and the phosphorylation of YLR190w was affected by the phosphate concentration, and the phosphorylation sites of YLR190w were Ser/Thr-Promotif, as revealed by protein mutation assay. In another library screen, YAF9, a yeast homolog of human AF9, was isolated using the two-hybrid system with YLR190w as the bait. It was revealed that interaction of YLR190w and YAF9 was affected by phosphate concentration. When all Ser/Thr in Ser/Thr-Pro motif were mutated to Ala, the interaction of YLR190w (mutant) and YAF9 was weakened, and the effect of phosphate concentration was impaired. Ylr190w was not involved in the PHO system by the acid phosphatase activity assay. Deletion of Ylr190w was constructed by homologous recombination and the doubling time of Ylr190w mutant strain as longer than that of wild type.

There are six ankyrin repeats in yeast transcriptional factor PHO81. The PHO81 ankyrin repeats fused with glutathione S-transferase (GST-ANK) was overexpressed in E. coli and it existed in inclusion body form. Using reduced and oxidized glutathione systems the fused protein fragment was denatured and renatured and the soluble protein was obtained. Then it was purified by affinity purification through glutathione sepharose column and its activity was analyzed. The PHO85-PHO80 and PHO85-PAP1 kinase complexes were prepared via coimmunoprecipitation respectively. With purified recombinant PHO4 protein as substrate of the kinase complexes when the purified GST-ANK was added the kinase activity of PHO85-PHO80 or PHO85-PAP1 kinase complex was inhibited. These studies indicate that the ankyrin repeats interact with PHO85-PHO80 or PHO85-PAP1 complex inhibit their kinase activities and are crucial in interaction between PHO81 and other proteins.

PHO4 and PHO2 protein were overexpressed in E. coli and purified respectively. The gel retardation assays showed that PHO4 and PHO2 protein could bind to -401 -289 bp sequence of PHO81 gene promoter respectively. The assays of the mutant PHO2 proteins where the Ser-230 was changed into Asp (GST-2SD) or Ala (GST-2SA) showed the binding patterns were the same as the intact PHO2 protein. Footprinting results located the PHO4 binding site at the 354 -333 bp sequence of PHO81 gene PHO2 binding site at the -341 -334 bp. Therefore the region (-401 -289 bp) contains the upstream activation sequence (UAS) of PHO81 gene and PHO4 protein cooperates with PHO2 in acting on the upstream sequence of PHO81 gene and both of them regulate the expression of PHO81 gene.

Deletion analysis on the fused PHO81-lacZ gene revealed two important regions in the upstream sequences of PHO81 gene -401 -289 bp and -1 012 -801 bp. They did not share higher similarity with the upstream regions of PHO5 and PHO84 gene, except that -401 -289 bp contains the 5'-CACGTG/T-3' motif, which was found among the upstream regions of PHO5 and PHO84 gene and was the core sequence of PHO4 binding site it also contains A/T-rich segments flanking the motif, which may be PHO2 binding sites. This suggests that the -401 -289 bp of the upstream region of PHO81 gene may contain upstream activation sequence (UAS), and -1 012 -801 bp may contain upstream enhancer sequence. The gel retardation assays of the -1 012 -801 bp was performed using yeast total protein extract, and the results showed that there was an unknown protein factor binding at the region.

Applying real time BIA(biomolecular interaction analysis) the interaction between yeast PHO4 and PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor chip via amino group. 5 &mgr;mol/L of recombinant PHO2 and PHO2 mutants which were fused with glutathione S-transferase were injected respectively. The mutant whose Ser 230 was changed into Asp (GST-2SD) showed high SPR (surface plasmon resonance) signal while wild type PHO2 and the mutant where Ser 230 was changed into Ala (GST-2SA) did not. This result indicated that there was interaction between PHO4 and GST-2SD proteins and the phosphorylation of the Ser 230 of PHO2 was essential. Five different concentration of GST-2SD (0.5 0.75 1.0 1.5 2.0&mgr;mol/L) were injected separately and the interaction kinetics was analyzed. The association rate constant is 2.4x10(4)(mol/L)(-1)s(-1) dissociation rate constant is 3.5x10(-5)s(-1) and association constant is 6.9x10(8) (mol/L)(-1).

No Abstract

Homology comparison of the novel PAP1 protein indicated that PAP1 protein is highly homologous to PHO85-associated protein PHO80, PCL1 and PCL2. We constructed the fused HA-PAP1 gene in frame for immunoprecipitation with anti-HA monoclonal antibody. Coimmunoprecipitation of fused HA-PAP1 and PHO85 protein, which were translated in vitro, verified the association of PAP1 with PHO85 protein. Simultaneously, we purified PHO4 protein expressed in E. coli and constructed a PHO85::TRP1 strain. After arranging the fused gene under the control of yeast ADH1 promoter, we transformed the resulting plasmids into yeast YPH499 and PHO85::TRP1 respectively, then prepared the yeast lysates for immunoprecipitation with anti-HA. We found that the immunoprecipitant complex of PAP1 can phosphorylate PHO4 protein in vitro, and the kinase activity is PHO85-associated, but is not related to CDC28 protein kinase. These data suggest that PAP1 is a putative cyclin and can form cyclin-CDK complex with PHO85 protein. Northern bolt with PAP1 gene as probe indicated there was no obvious difference during the different phase of the cell-cycle in the transcription of PAP1 gene. We constructed the PAP1::TRP1 strain, and the rAPase activity analysis showed PAP1 had no function in the PHO system.

p53 interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function involving cell cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression. To identify these proteins, we used the yeast two-hybrid system and screened a HeLa cDNA library. Six positive colonies were isolated from 1.5x10(6) transformants. The cDNA sequence of each positive colony was determined. Two novel cDNA fragments (p53BP1 and p53BP2) were cloned. These two cDNA fragments code for the same protein composed of 158 amino acids, which shows high similarity to the ubiquitin-conjugating enzyme (UBC9) of H. sapiens as well as to E2s from other organisms, such as UBC (76 %) of C. elegans, HUS5(66 %) of S. pombe, UBC(66 %) of A. thaliana and UBC9(56 %) of S. cerevisiae. A cDNA fragment from p53BP1 was used to probe a Northern blot containing poly(A)(+) RNA from various human tissues and various cell lines. At high stringency this probe hybridized to a single mRNA of approximately 1.2 kb that was expressed in heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, human cervical carcinoma cell (HeLa), human mammary carcinoma cell (MCF-7), human lymphoma cell (Jurkatt) and human teratocarcinoma cell (PA-I). It is not expressed in brain, lung, human lung carcinoma cell, human heptocellular carcinoma cell (HepG2) and human glioma cell(U251MG).

Yeast regulatory factor PHO85, which is a cyclin-dependent kinase (DCK), participates in the regulation of the cell cycle and the expression of the acid phosphatase gene. Using PHO85 as target, we have cloned from the yeast two-hybrid genomic library a novel gene (PAP1) with product which can asscociate with the PHO85 protein. The PAP1 gene including the 5apo; and 3apo; flanking sequence was cloned and sequenced. It codes for a protein composed of 284 amino acids, which contains a cyclin conservative region (150-263 aa), and shares 63% similarity with the conservative region of PHO80. The N-terminal of the PAP1 protein abounds in PEST sequence, which is believed to be common to rapidly degraded proteins. Using the yeast two-hybrid system, we demonstrated that both the full-length PAP1 protein and the C-terminal (99-285 aa) can interact with PHO85. The coding sequence of the PAP1 gene was cloned and sequenced, then arranged under the control of the P(L) promoter, and expressed in E. coli BL21 (DE3) pLysS. After heat induction at 42 degrees, protein band of approximately 32 kD was observed in the SDS-PAGE, which fits the molecular size of the hypothetical PAP1 protein. The 10 N-terminal amino acids of the 32 kD protein agreed with the deduced PAP1 amino acid sequence. The protein was found as inclusion body in E. coli, and accounted for 50% of the precipitant after purification.

Both PHO2 and PHO4 are positive regulatory factors of yeast PHO5 gene. Here we show that the PHO2 fused to yeast transcriptional factor GAL4 DNA-binding domain activates the expression of the reporter gene (lacZ), and the lacZ activities were regulated by Pi concentration, therefore it could be suggested that there are acidic activation domains on the PHO2 protein. Acidic amino acid rich region of 287-326 aa in PHO2 is not a transcriptional activation domain. PHO2 maintained its activation activity only if Ser230 is phosphorylated, thus the phosphorylated site may play a key role in the transcriptional activation function of PHO2. PHO4 fused to the GAL4 DNA-binding domain also activates the expression of lacZ. A segment of 1-97 aa at its N-terminal is responsible for the transcriptional activation activity. A two-hybrid assay reveals that there exists interaction between PHO2 and PHO4 protein, and the interaction affects their transcriptional activation function.

It is found that minor changes around the basic motif (88-160) and the acidic motif (771-810) of the PHO81 protein can lead to the constitutive expression of the acid phosphatase gene (PHO5), and the two motifs work cooperatively. The PHO81 protein has six ankyrin repeats, which are the recognition sites of PHO81 protein with the PHO80-PHO85 protein complex. It is found that the ankyrin repeats 1,2,4,5 and 6 are important for the PHO81 protein, but the deletion of Pro(509) and Leu(510) in ankyrin repeat 3 does not affect the PHO81 protein. We have found a candidate nucleoplasmin-like nuclear location sequence at 701-719 of the PHO81 protein and the deletion of the corresponding DNA fragment inactivates the PHO81 protein. However, when the conservative amino acids Arg701 and Lys702 are constituted by Ser and Gln, or Lys717 replaced by Asn, or Arg719 is turned to Ser, the function PHO81 protein is not affected.

Yeast PHO2 protein plays a role in the expression of several different genes and acts as a multiple global activator. Here we report the comparison of the effect of PHO2 protein on the expression of PHO5, HIS4 and Ho genes. In the PHO2 defective yeast strain, PHO5 gene could not be depressed in low Pi and the expression of the HIS4 and HO genes was decrease to 25% and 40% of the normal level respectively. When the PHO2 gene carried by low copy shuttle vector was transformed into this strain, the expression of the three genes could be restored. Previously the PHO2 gene was mutated on its conspicuous regions with site-directed mutagenesis, deletion and linker insertion. Here the effects of these mutations on these genes were compared.

The ciliary neurotrophic factor (CNTF) plays a very important role in the development and regeneration of the nervous system. In this study, the prediction of secondary structure and the hydrophobicity analysis of human CNTF were performed according to the amino acid sequence deduced from the nucleotide sequence of the cDNA. Based on the results of the prediction of structure, the human CNTF gene was modified by insertion and deletion mutagenesis. The various mutants were all highly expressed in E. coli. The recombinant proteins were purified from bacterial via DEAE A-50 and Sephacryl S-200 chromatography, and their survival-promoting activities were determined by using cultures of the dorsal root ganglion neurons of embryonic chick. The results showed that the alpha-helixes in CNTF were critical for the biological activity and the flexible C-terminus of human CNTF was not essential. Our data also indicated that the middle and the tail part of the D-helix might play crucial roles in the biological functions of CNTF.

The GST gene fusion system was used to express PHO2 gene and its mutants in E. coli Gel retardation assays showed that PHO2 fusion protein can bind to the upstream activation sequence (UAS) of the acid phosphatase gene PHO5. The homeodomain of PHO2 protein has such structure as alpha-helix 2-beta-turn-alpha-helix 3, which acts as the DNA binding domain of the transcriptional factor. The Mutation of lle 123 to Pro in helix 3 or the insertion of 4 amino acids (PDPD) between 112 and 113 in alpha-helix 2 led to the complete loss of DNA-binding activity of PHO2, while the mutation of Pro 117 to Ala in beta-turn did not affect the binding activity significantly. Deletions of the PHO80 homologous region, acidic region or C-terminal 132 residues had no great effect on the DNA-binding activity, although these mutants had lost the ability to activate PHO5 expression in vivo.

We report here that PHO2 protein is also phosphorylated by an unidentified protein kinase. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cc2/CDC28-related kinase in the PHO2 protein led to the complete loss of its ability to activate the transcription of PHO5 gene. Further work showed that Pro-231 to Ser mutation inactivated PHO2 protein as well, while Ser-230 to Asp mutation did not affect PHO2 activity. Since PHO2 Asp-230 mutant mimics Ser-230 phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of the PHO5 gene. The results of in vitro phospho-labelling experiments showed that the whole cell extract of the YPH499 strain grown under low phosphate conditions phosphorylated GST (glutathione S-transferase)- PHO2 (wild type) fusion protein, but not the GST-PHO2 mutant (Pro-231 to Ser) protein in which the putative phosphorylation sequence was destroyed. We therefore propose that the PHO2 protein may also be phosphorylated in vitro at Ser-230, and the phosphorylation of this site may be necessary for its function in controlling PHO5 gene expression.