Final Activity Report Summary - PHOSPHOGENOMICS (New approaches to the study of protein phosphorylation: a postgenomic view)
In order to identify new mechanisms of protein regulation by phosphorylation, we purified a set of yeast protein kinases, expressed as a GST- fusion proteins, in a pep4 yeast strain. One of the substrates studied was the essential yeast type 1 protein phosphatase (PP1) inhibitor Ypi1. The importance of the Ypi1 phosphorylation is was object of the study.
We also identified potential substrates for the yeast MAP kinase Slt2 by using the yeast proteome chip. Between the potential candidates we found transcription factors, regulators of the Slt2 MAP kinase pathway, cytoskeletal proteins, etc. We analysed the changes in the expression pattern that the lack of Ypi1 induced. Ypi1, was expressed under control of a doxycyclin-repressible promoter. The most significant result in the absence of Ypi1 was a very fast decrease of the CDC26 mRNA levels. Sds22, another essential PP1 regulatory subunit, forms a complex with Ypi1. We used a conditional temperature sensitive mutant to study the changes in the expression profiles that the shift to the restrictive temperature induces and demonstrated that several genes typically expressed in haploid cells were inhibited in the absence of a functional Sds22.
Using DNA microarrays experiments we have determined the contribution of the Slt2 MAPK to the transcriptional response elicited by alkali stress in yeast. Several proteins are specifically transcribed in a Slt2-dependent manner when the pH of the culture media increases. Interestingly, all are cell wall-related proteins, involved in remodelling the cell wall structure. We also identified the cell wall protein responsible for the sensing of high pH stress.
The family of PP type 2C in yeast comprises a family of proteins whose functions are not completely understood. We analysed the expression changes that the lack of each single member of the family (Ptc1-5) provoked. We showed that Ptc1 has a specific and differentiated role in the cell and that the lack of this protein triggers a cell wall damage response.
The pair of the redundant PK (Pkh1 and Pkh2) structurally related with the human PDK1 was also objective of our studies. Since the lack of both PK is lethal we made a pkh1 strain were the expression of PKH2 is under control of the doxycyclin-repressible promoter. The lack of the Pkh function has several effects, including the regulation of the expression of a gene coding for a protein involved in the metabolism of a lipid required for Pkh activity regulation.
We also identified potential substrates for the yeast MAP kinase Slt2 by using the yeast proteome chip. Between the potential candidates we found transcription factors, regulators of the Slt2 MAP kinase pathway, cytoskeletal proteins, etc. We analysed the changes in the expression pattern that the lack of Ypi1 induced. Ypi1, was expressed under control of a doxycyclin-repressible promoter. The most significant result in the absence of Ypi1 was a very fast decrease of the CDC26 mRNA levels. Sds22, another essential PP1 regulatory subunit, forms a complex with Ypi1. We used a conditional temperature sensitive mutant to study the changes in the expression profiles that the shift to the restrictive temperature induces and demonstrated that several genes typically expressed in haploid cells were inhibited in the absence of a functional Sds22.
Using DNA microarrays experiments we have determined the contribution of the Slt2 MAPK to the transcriptional response elicited by alkali stress in yeast. Several proteins are specifically transcribed in a Slt2-dependent manner when the pH of the culture media increases. Interestingly, all are cell wall-related proteins, involved in remodelling the cell wall structure. We also identified the cell wall protein responsible for the sensing of high pH stress.
The family of PP type 2C in yeast comprises a family of proteins whose functions are not completely understood. We analysed the expression changes that the lack of each single member of the family (Ptc1-5) provoked. We showed that Ptc1 has a specific and differentiated role in the cell and that the lack of this protein triggers a cell wall damage response.
The pair of the redundant PK (Pkh1 and Pkh2) structurally related with the human PDK1 was also objective of our studies. Since the lack of both PK is lethal we made a pkh1 strain were the expression of PKH2 is under control of the doxycyclin-repressible promoter. The lack of the Pkh function has several effects, including the regulation of the expression of a gene coding for a protein involved in the metabolism of a lipid required for Pkh activity regulation.