title: Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.
abstract: Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: zaneta@ibb.waw.pl.
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Jastrzebska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: kaminska@ibb.waw.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kaminska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: chelstowska@ibb.waw.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Chelstowska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: adomania@o2.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Domanska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: wr@ibb.waw.pl.
authors.forename: Weronika
authors.initials: W
authors.lastname: Rzepnikowska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: ewa@ibb.waw.pl.
authors.forename: Ewa
authors.initials: E
authors.lastname: Sitkiewicz
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: piotr.cholbinski@insp.waw.pl.
authors.forename: Piotr
authors.initials: P
authors.lastname: Cholbinski
authors.affiliation: Kent Fungal Group, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK. Electronic address: C.W.Gourlay@kent.ac.uk.
authors.forename: Campbell
authors.initials: C
authors.lastname: Gourlay
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: danka@ibb.waw.pl.
authors.forename: Danuta
authors.initials: D
authors.lastname: Plochocka
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. Electronic address: teresa@ibb.waw.pl.
authors.forename: Teresa
authors.initials: T
authors.lastname: Zoladek
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D043743:Ubiquitin-Protein Ligase Complexes; D017868:Cyclic AMP-Dependent Protein Kinases; C469287:RSP5 protein, S cerevisiae
keywords: Phosphorylation; Protein kinase A; Rsp5 Ubiquitin ligase; Yeast