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The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.

ABSTRACT: We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing 32P-labeled orthophosphate. The purified 32P-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies. Furthermore, trypsin treatment of the 32P-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo. Incubation of phosphorylated peptides with calf intestinal phosphatase liberated the radiolabel as free phosphate, indicating a phosphoester linkage. Acid hydrolysis of the tryptic peptides revealed 32P-label label comigrating with phosphoserine; some of it, however, was also identified as phosphothreonine. Using anti-OBF1 antibodies, we cloned the OBF1 gene from a lambda gt11 yeast expression library. The DNA sequence of the isolated gene and its over-expression in yeast indicated that OBF1 is identical to ABF-1 and BAF1 proteins, believed to have a role in transcriptional repression and activation. Therefore, we suggest that OBF1 is a multifunctional protein, acting in transcription and replication, and that these activities are regulated by phosphorylation.

SUBMITTER: Francesconi SC

PROVIDER: S-EPMC51603 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.

Francesconi S C SC Eisenberg S S

Proceedings of the National Academy of Sciences of the United States of America 19910501 10

We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing 32P-labeled orthophosphate. The purified 32P-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies. Furthermore, trypsin treatment of the 32P-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo. Incubation of phosphorylated peptides with calf intestinal phospha ...[more]

PMID: 2034654

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Project description:Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis.Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the United States, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps.

Project description:The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (V(max)) values of the full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7+/-74.8 (Ser-164) and 34. 4+/-2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

Dataset Information

The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.

Publications

The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.

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